A method for preparing a polypeptide or protein-polymer conjugate by a heavy metal-free initiation polymerization technique

Abstract

This invention discloses a method for preparing peptide or protein-polymer conjugates using a heavy metal-free initiation polymerization technique. The method comprises two steps: the first step involves reacting the peptide with a compound containing vinyl sulfone (VS) groups under specific conditions; the second step involves adding a zwitterionic monomer under the initiation of an organic base to generate a zwitterionic polymer chain. This achieves site-specific coupling of the peptide's thiol groups and yields stable peptide or protein-polymer conjugates. This method provides a polymerization approach that "grows" polymer chains at specific sites on the peptide. Furthermore, this method is environmentally friendly, as it does not involve the addition of Cu. + Using metal ions, the resulting polypeptides or protein-polymer conjugates have stable structures, making it a promising and widely applicable method.

Description

Technical Field

[0001] This invention relates to a method for the specific and efficient modification of polypeptides or proteins containing thiol sites by a compound containing a vinyl sulfone (VS) group in solution, and the polymerization of the compound into a polymer chain initiated by the vinyl sulfone (VS) group at the other end. Technical Background

[0002] In recent years, the biopharmaceutical industry has developed rapidly. Compared with traditional small molecule chemical drugs, therapeutic protein drugs (biopharmaceuticals) have greater advantages in treatment, such as higher target specificity and pharmacological efficacy. However, ordinary protein drugs suffer from problems such as short half-life, high clearance rate, large molecular weight, poor permeability, susceptibility to degradation by enzymes, bacteria, and body fluids, and low bioavailability of non-injectable administration methods. To address these issues, the concept of long-acting protein drugs has been proposed, which involves increasing the half-life of protein drugs to achieve a longer therapeutic effect. Methods for achieving long-acting protein drugs have been a hot research topic.

[0003] The chemical methods for long-acting protein drugs generally involve chemically coupling macromolecules and protein drugs. Coupling methods are divided into non-site-specific coupling and site-specific coupling. Non-site-specific coupling involves non-specific modification of random sites, while site-specific coupling involves specific modification of a specific site. Currently, most methods for coupling protein drugs and polymers are non-site-specific couplings. However, random site modification not only fails to yield homogeneous products and makes subsequent separation and purification difficult, resulting in low yields, but it can also mask the active sites of the protein drug, affecting the bioactivity of the coupled drug. Therefore, non-site-specific coupling methods have significant drawbacks. To address these issues, site-specific coupling methods have emerged. However, these methods are currently imperfect. For example, the traditional ATRP (autoradical polymerization) site-specific coupling method typically introduces a large amount of Cu+ ions, which are difficult to remove. Copper, as a heavy metal element, not only poses certain health risks but also affects further operations. Moreover, these methods are generally costly, lack broad applicability, and result in structurally unstable products, with polymers easily detaching, affecting the bioactivity of the coupled drug. There is an urgent need for a site-directed coupling method that is simple to operate, low in cost, environmentally friendly, broadly applicable, and provides stable drug structures. Summary of the Invention

[0004] To address the aforementioned problems, this invention uses peptides or proteins as model compounds and aims to provide a method for preparing peptide or protein-polymer conjugates using heavy metal-free initiation polymerization technology. This novel polymerization method "grows" polymer chains at specific sites of the peptide. The method is highly specific, environmentally friendly, and Cu-free. + The participation of this method results in stable structures of peptides or protein-polymer conjugates, making it a promising and widely applicable approach.

[0005] A method for preparing peptide or protein-polymer conjugates using heavy metal-free initiation polymerization, the method comprising the following steps:

[0006] Step 1: Dissolve the polypeptide or protein containing thiol group in 20-80% ethanol or acetonitrile solution, add a compound containing vinyl sulfone group (VS), react at room temperature for 1-12 hours, then extract and purify by layering to obtain the supernatant.

[0007] Step 2: Under the initiation of organic base, adjust the pH of the supernatant obtained in step (1) to 7-10, and the zwitterionic monomer undergoes anionic polymerization. The reaction is carried out at -20 to 50℃ for 1 to 24 hours to generate zwitterionic polymer chains. This achieves site-directed coupling of thiol groups and obtains stable polypeptide-polymer conjugates.

[0008] Compounds containing a vinyl sulfone group are selected from divinyl sulfone (structure I below) and diacetylsulfonylmethane (structure II below).

[0009]

[0010] In the preferred embodiment of the above-described technical solution, the thiol-containing polypeptide may be glutathione (GSH) or the like, and the thiol-containing protein may be a protein with a thiol group at its C-terminus or N-terminus, such as interferon α2b.

[0011] In the above-described technical solution, in a preferred embodiment, the organic base is an organic compound containing an imidazole group, such as imidazole, 1-vinylimidazolium, 1-methylimidazolium, etc., preferably 1-methylimidazolium.

[0012] In the preferred embodiment of the above-described technical solution, the zwitterionic monomer is selected from methacrylic acid sulfonate betaine (SBMA) and methacrylic acid carboxylic acid betaine (CBMA).

[0013] In the above-described technical solution, in a preferred case, the first step reaction does not require pH adjustment, and the second step reaction is adjusted to pH 7-10, preferably pH 7-8.

[0014] In the above-described technical solution, in a preferred embodiment, the molar ratio of the compound containing sulfone groups at both ends to the polypeptide or protein containing thiol groups in the first step is 1:5 to 1:100, preferably 1:10 to 1:40; and in the second step, the molar ratio of the product from the previous step to the zwitterionic monomer is 1:10 to 1:100, preferably 1:40 to 1:60.

[0015] In the preferred embodiment of the above-described technical solution, the amount of the organic base used is 1% to 5% of the molar concentration of the first step product.

[0016] In the preferred embodiment of the above-described technical solution, the temperature of the second reaction step is 20℃~40℃.

[0017] In the preferred embodiment of the above-described technical solution, the reaction time of the first step is 3-6 hours; the reaction time of the second step is 3-6 hours.

[0018] In the preferred embodiment of the above-described technical solution, the specific operation of extraction and purification in the first step is as follows: remove excess ethanol or acetonitrile by rotary evaporation, then add dichloromethane for extraction, and take the supernatant after standing and separating the layers.

[0019] Beneficial effects:

[0020] The zwitterionic modification method for polypeptides or proteins described in this invention has the following advantages over traditional methods:

[0021] (1) It has a wide substrate applicability and is applicable to a variety of peptides or proteins containing thiol groups.

[0022] (2) It has broad applicability and can modify various zwitterionic polymers;

[0023] (3) The reaction conditions are mild, without high temperature, low temperature, strong acid, strong alkali or other environments;

[0024] (4) It has high specificity, conforms to atomic economics, is green and environmentally friendly, and does not involve metal ions such as Cu+.

[0025] (5) It has good stability and the generated polypeptides or protein-polymer complexes are not easily decomposed and detached.

[0026] In summary, this method is a site-directed coupling method that is simple to operate, low in cost, environmentally friendly, widely applicable, and produces stable drug structures. Attached Figure Description

[0027] Figure 1 GSH-VS was characterized by proton nuclear magnetic resonance spectroscopy.

[0028] Figure 2 GSH-VS-PSBMA was characterized by 1H NMR spectroscopy under different pH conditions.

[0029] Figure 3 GSH-VS-PSBMA was characterized by proton NMR spectroscopy under different temperature conditions.

[0030] Figure 4 GSH-VS-PSBMA was characterized by 1H NMR spectroscopy under different reaction time conditions.

[0031] Figure 5 : Hydrogen NMR spectroscopy characterization of GSH-BVS-PSBMA.

[0032] Figure 6 : Hydrogen NMR spectroscopy characterizes interferon α2b-VS-PSBMA.

[0033] In the figure: ppm=4.7 is the characteristic peak of deuterium water, and ppm=5.4 is the characteristic peak of dichloromethane. Detailed Implementation

[0034] This invention provides a method for the specific and efficient modification of peptides containing thiol sites in solution. The method involves dissolving the thiol-containing peptide or protein in a 50% ethanol solution, dispersing it evenly, adding a compound containing a vinyl sulfone group, and reacting at room temperature for 1-12 hours. The obtained product is rotary evaporated to remove excess ethanol, then extracted with dichloromethane, centrifuged, allowed to stand, and the supernatant is collected. An amphoteric monomer and an organic base are added to the supernatant to adjust the pH to 7-10, and the reaction is carried out at -20-50°C for 1-12 hours to obtain the final product. The compound containing a vinyl sulfone group is selected from divinyl sulfone and diacetylsulfonylmethane; the organic base is an organic compound containing an imidazole group, such as imidazole, 1-vinylimidazolium, 1-methylimidazolium, etc., preferably 1-methylimidazolium.

[0035] The following specific embodiments are for further explanation of the content of the present invention and should not be construed as limiting the present invention in any way.

[0036] Example 1: pH screening and characterization of the synthesized GSH-VS-PSBMA (glutathione-coupled polymethacrylate sulfonate betaine containing vinyl sulfone groups).

[0037] Glutathione (0.01 g) was dissolved in 8 ml of 50% ethanol (v / v) solution and dispersed evenly. 100 μL of DVS (vinyl sulfone) was added (DVS was in significant excess), and the mixture was reacted at room temperature for 3 h. The resulting product was vacuum rotary evaporated to remove ethanol, and then extracted with dichloromethane. After centrifugation and separation, the supernatant was collected. 3 ml of the supernatant was divided into three test tubes, 1 ml in each. SBMA (methacrylic acid sulfonate betaine) was added to each tube, with a molar ratio of SBMA to GSH-VS in the supernatant of approximately 30:1. An organic base, 1-methylimidazole, was then added to adjust the pH of the solutions in the three test tubes to 7, 8, and 9, respectively. The three test tubes were then reacted at 37 °C for 6 h to obtain the final product. The NMR characterization results of the first step in the synthesis of GSH-VS are as follows: Figure 1 As shown, the NMR characterization of the final synthesized product GSH-VS-PSBMA is as follows: Figure 2As shown in the figure, the chemical shifts of the final product GSH-VS-PSBMA are characteristic peaks of the methyl group after chain formation at 1.1–1.2 ppm (as shown in a), and characteristic peaks of the methylene group after chain formation at around 1.8 ppm (as shown in b), proving that the reaction was successful.

[0038] Example 2: Temperature screening and characterization of the synthesis of GSH-VS-PSBMA (glutathione-coupled polymethacrylate sulfonate betaine containing vinyl sulfone groups)

[0039] Reduced glutathione (0.015 g) was dissolved in 11 ml of 50% ethanol (v / v) solution and dispersed evenly. 100 μL of DVS (vinyl sulfone) was added (DVS in large excess), and the reaction was carried out at room temperature for 3 h. The obtained product was vacuum rotary evaporated to remove ethanol, and then extracted with dichloromethane. After centrifugation and separation, the supernatant was collected. 5 ml of the supernatant was divided into 5 test tubes, 1 ml in each. SBMA (methacrylic acid sulfonate betaine) was added to each tube, with a molar ratio of SBMA to GSH-VS in the supernatant of approximately 30:1. An organic base, 1-methylimidazole, was added to adjust the pH of the solution in the 5 test tubes to 8, approximately equal to the pH of GSH-VS. The 5 test tubes were then reacted at -20℃, 4℃, 25℃, 37℃, and 50℃ for 6 h, respectively, to obtain the final product. The NMR characterization of the final product GSH-VS-PSBMA is as follows: Figure 3 As shown in Figure 1, the chemical shifts of the final product GSH-VS-PSBMA are characteristic peaks of the methyl group after chain formation at 1.1–1.2 ppm (Figure 1), and around 1.8 ppm is the characteristic peak of the methylene group after chain formation (Figure 2), proving the reaction was successful. Using the GSH characteristic peak as a baseline, integral calculations were performed, with the ordinate representing the peak area, indicating the amount of SBMA monomers polymerized. This demonstrates the success of the reaction, and that temperature has little effect on the anionic polymerization reaction.

[0040] Example 3: Time screening and characterization of the synthesis of GSH-VS-PSBMA (glutathione-coupled polymethacrylate sulfonate betaine containing vinyl sulfone groups)

[0041] Glutathione (0.012 g) was dissolved in 9 ml of 50% ethanol (v / v) solution and dispersed evenly. 100 μL of DVS (vinyl sulfone) was added (DVS in large excess), and the reaction was carried out at room temperature for 3 h. The obtained product was vacuum rotary evaporated to remove ethanol, and then extracted with dichloromethane. After centrifugation and separation, the supernatant was collected. 4 ml of the supernatant was divided into four test tubes, 1 ml in each. SBMA (methacrylic acid sulfonate betaine) was added to each tube, with a molar ratio of SBMA to GSH-VS in the supernatant of approximately 30:1. An organic base, 1-methylimidazole, was added to adjust the pH of the solutions in the four test tubes to 8. The four test tubes were then reacted at 37 °C for 15 min, 1.5 h, and 3 h, respectively. 10 μL of DCl was added as a quencher to obtain the final product. The NMR characterization of the final product GSH-VS-PSBMA is as follows: Figure 4 As shown in the figure, the chemical shifts of the final product GSH-VS-PSBMA are as follows: 1.1–1.2 ppm is the characteristic peak of the methyl group after chain formation (shown in a), around 1.8 ppm is the characteristic peak of the methylene group after chain formation (shown in b), and around 3.8 ppm is the characteristic peak of deuterated hydrochloric acid, proving that the reaction was successful and proceeded very rapidly.

[0042] Example 4: Characterization of the synthesized GSH-BVS-PSBMA (glutathione coupled with polymethacrylate sulfonate betaine containing diacetyl sulfomethane)

[0043] Glutathione (0.01g) was dissolved in 9ml of 20% acetonitrile (v / v) solution and dispersed evenly. 30mg of BVS (bis(acetyl)sulfonylmethane) was added (BVS was in significant excess), and the reaction was carried out at room temperature for 3 hours. The resulting product was vacuum rotary evaporated to remove ethanol, and then extracted with dichloromethane. After centrifugation and separation, the supernatant was collected. 1ml of the supernatant was placed in a test tube, and SBMA (methacrylic acid sulfonate betaine) was added. The molar ratio of SBMA to GSH-BVS in the supernatant was approximately 30:1. An organic base, 1-methylimidazole, was added to adjust the pH of the solution in the test tube to 8. The reaction was then carried out at 37℃ for 3 hours to obtain the final product. The NMR characterization of the final product, GSH-BVS-PSBMA, is as follows: Figure 5 As shown in the figure, the chemical shifts of the final product GSH-BVS-PSBMA are 1.1–1.2 ppm, which is the characteristic peak of the methyl group after chain formation (shown in a), and around 1.8 ppm, which is the characteristic peak of the methylene group after chain formation (shown in b), proving that the reaction was successful.

[0044] Example 5: Characterization of the synthesized interferon α2b-VS-PSBMA (interferon α2b containing vinyl sulfone groups coupled with polymethacrylate sulfonate betaine)

[0045] Recombinant human interferon α2b (0.01 g) was dissolved in 9 ml of 50% ethanol (v / v) solution and dispersed evenly. 100 μl of DVS (vinyl sulfone) was added (DVS in large excess), and the reaction was carried out at room temperature for 3 h. The obtained product was vacuum rotary evaporated to remove ethanol, and then extracted with dichloromethane. After centrifugation and separation, the supernatant was collected. 2 ml of the supernatant was placed into test tubes, and SBMA (methacrylic acid sulfonate betaine) was added. The molar ratio of SBMA to interferon α2b-VS in the supernatant was approximately 30:1. An organic base, 1-methylimidazole, was added to adjust the pH of the solution in the test tubes to 8. The reaction was carried out at 37 °C for 3 h to obtain the final product. The NMR characterization of the final synthetic product, interferon α2b-VS-PSBMA, is as follows: Figure 6 As shown in the figure, the chemical shifts of the final product, interferon α2b-VS-PSBMA, are characteristic peaks of the methyl group after chain formation at 1.1–1.2 ppm (as shown in a), and around 1.8 ppm is the characteristic peak of the methylene group after chain formation (as shown in b), proving that the reaction was successful.

Claims

1. A method for preparing a polypeptide or protein-polymer conjugate by a polymerization technique initiated without involvement of heavy metals, characterized in that, The method comprises the following steps: The first step: dissolve the polypeptide or protein containing sulfydryl in 20-80% volume fraction of ethanol or acetonitrile solution, add the compound containing ethylenesulfone group, react at normal temperature for 1-12 hours, then extract and purify to obtain supernatant; The second step: add the zwitterionic monomer to the supernatant obtained in the first step, initiate under the organic base, adjust the pH value of the supernatant to 7-10, the zwitterionic monomer occurs anionic polymerization, react at-20-50℃ for 1-24 hours to generate zwitterionic polymer chain; The compound containing ethylenesulfone group is selected from diethylenesulfone and bisacetyl sulfomethane; The polypeptide containing sulfydryl is glutathione; the protein containing sulfydryl is interferon α2b; The zwitterionic monomer is selected from methacrylic acid sulfobetaine and methacrylic acid carboxybetaine; The organic base is the organic compound containing imidazole group; The molar ratio of the compound containing ethylenesulfone group to the polypeptide or protein containing sulfydryl in the first step is 1:5-1:100; the molar ratio of the product of the previous step to the zwitterionic monomer in the second step is 1:10-1:

100.

2. The method of claim 1, wherein, The molar ratio of the compound containing ethylenesulfone group to the polypeptide or protein containing sulfydryl in the first step is 1:10-1:40; the molar ratio of the product of the previous step to the zwitterionic monomer in the second step is 1:40-1:

60.

3. The method of claim 1, wherein, The organic base is imidazole, 1-vinylimidazole and 1-methylimidazole.

4. The method of claim 1, wherein, The amount of the organic base is 1%-5% of the molar concentration of the product of the first step.

5. The method of claim 1, wherein, The reaction time of the first step is 3-6 hours; the reaction temperature of the second step is 20-40℃, and the reaction time of the second step is 3-6 hours.

6. The polypeptide or protein-polymer conjugate prepared by the method of claim 1.

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