Preparation method and activity evaluation of PEG modified medicinal polypeptide with different connecting arm numbers

Abstract

The invention belongs to the technical field of biological medicines, and provides a preparation method and activity evaluation of PEG modified medicinal polypeptides with different connecting arm numbers, and the selected medicinal polypeptides are anti-tumor polypeptides and antibacterial peptides with toxic and side effects, such as toxin peptides and the like; pegylation (PEG) of different connecting arm numbers is carried out on the tail end of the medicinal polypeptide; different numbers of PEG connecting arms are connected to the hydrophobic end of the toxin peptide through solid-phase synthesis, operation is easy, and preparation is easy; the PEG modified by different numbers of connecting arms can effectively regulate and control the whole body toxic and side effects of the toxin peptide, the toxicity of the toxin peptide is gradually reduced along with the increase of the number of the PEG connecting arms, the biosafety of the toxin peptide in an effective toxicity range is favorably improved, the circulation time of the toxin peptide in a body is prolonged, and the killing capability of the toxin peptide on normal tissues and cells is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically is a preparation method and activity evaluation of PEG-modified medicinal polypeptides with different numbers of connecting arms. Background Art

[0002] Peptides are a class of compounds formed by connecting multiple amino acids through peptide bonds. Compared with small molecule drugs, peptides have strong affinity with targets, high specificity, and few side effects; compared with macromolecule drugs, peptide drugs have obvious advantages such as low immunogenicity and low production cost. However, the disadvantages of peptide drugs are also obvious. The physical and chemical properties of peptide drugs are unstable, they are easily oxidized and hydrolyzed, they are prone to aggregation, they have a short half-life, and they have a fast clearance rate.

[0003] Toxin peptides are a class of toxic natural polypeptide products, mostly derived from poisonous animals, such as bees, spiders, scorpions, snakes, etc., and are often used in biomedical fields such as anti-tumor, antibacterial and antiviral. Although there are many types of toxin peptides, there are still relatively few types of peptides developed for treatment, mainly due to the problems of poor pharmacokinetic stability, high immunogenicity and low bioavailability of toxin peptides. In particular, the toxicity of a single toxin peptide to cells is too high. While exerting anti-tumor, antibacterial and antiviral effects, it indiscriminately attacks and kills abnormal and normal cells, often causing systemic toxic side effects, which limits its practical clinical application.

[0004] Chemical modification is a common method to improve the performance of peptide molecules, including terminal modification, residue modification and backbone modification. Among them, terminal modification can consider introducing functional groups at the nitrogen and carbon ends for structural optimization and performance modification, which is of great significance in enhancing the pharmacokinetic stability and prolonging the half-life of peptides. Polyethylene glycol (PEG) is a polymer with high stability and good biocompatibility. It is widely used in the terminal modification of proteins and peptides. It can effectively prolong the half-life of proteins and peptides, reduce or eliminate immunogenicity, reduce toxic side effects, and improve their chemical and biological stability.

[0005] Since pure toxin peptides have many limitations such as high cytotoxicity and easy degradation in the body, a PEG modification method of connecting different numbers of arms to the end of the toxin peptide has been proposed. The good stability and biocompatibility of PEG can be used to improve the in vivo stability and pharmacokinetic properties of toxin peptides, effectively reduce their toxicity to cells, reduce side effects in the body, and expand the application range of toxin peptides. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a preparation method and activity evaluation of PEG-modified medicinal polypeptides with different numbers of linkers, so as to solve the shortcomings of poor biological stability and large systemic toxic side effects of existing toxin peptides, and provides a synthesis method for modifying the terminal of toxin peptides with PEG with different numbers of linkers, thereby improving the pharmacokinetic stability of toxin peptides, reducing the toxicity of toxin peptides to cells, and helping to promote the clinical application of bee venom peptides.

[0007] According to the first aspect of the present disclosure, a method for preparing PEG-modified pharmaceutical polypeptides with different numbers of linkers is proposed, comprising the following steps:

[0008] S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, then removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM; diisopropylethylamine DIEA, dimethylformamide DMF, DCM, nitrogen bubbling reaction for 60 minutes; adding methanol, reacting for half an hour, removing the reaction solution, and washing with DMF and MEOH;

[0009] S2, add the second amino acid 2n in the sequence, 1-hydroxyl, benzotriazole tetramethyl hexafluorophosphate HBTU and DIEA to the reactor, and react with N2 bubbling for half an hour, wash off the liquid, and detect with ninhydrin; then cap with pyridine and acetic anhydride; wash, add decapping liquid to remove the 9-fluorenylmethoxycarbonyl Fmoc protecting group, wash, and detect with ninhydrin;

[0010] S3, adding different amino acids in the sequence in sequence and modifying them according to step S2;

[0011] S4. After the resin is dried with nitrogen, it is removed from the reaction column and poured into a flask. Then, a cutting solution is added to the flask, shaken, and the resin is filtered out. The filtrate is obtained, and ether is added to the filtrate to precipitate the crude product, which is then centrifuged and washed to obtain the crude product of the sequence. The crude product is purified by high performance liquid chromatography, and the successful synthesis of the modified peptide is determined by mass spectrometry.

[0012] Preferably, in step S1, when the first amino acid of 2n in the sequence is added, and 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM are added, the amounts of dimethylformamide DMF and DCM are such that the resin can be fully agitated.

[0013] Preferably, the amount of the cutting liquid added in S4 is added in a ratio of 10 mL / g to the resin.

[0014] Preferably, the cutting fluid is composed of 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water.

[0015] According to the second aspect of the present disclosure, a method for evaluating the activity of medicinal polypeptides modified with PEG with different numbers of linkers is proposed, wherein the medicinal polypeptides are anti-tumor polypeptides and antimicrobial peptides with toxic side effects, including toxin peptides; different numbers of PEG linkers are modified at the N-terminus of the toxin peptide by solid phase synthesis, ultimately forming toxin peptide chains with different types of chemical modifications.

[0016] Preferably, the number of PEG arms of the polypeptide modified chain is 2-16.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention synthesizes toxin peptides modified with different numbers of PEG linkers based on polypeptide solid phase synthesis technology, which is simple to operate and easy to produce.

[0019] 2. The toxin peptide polypeptide drug prepared by the present invention is modified with PEG with different numbers of connecting arms at the N-terminus to improve the stability of the toxin peptide in vivo; wherein the N-terminal modified PEG can effectively reduce the toxicity of the toxin peptide to cells, and as the number of PEG connecting arms increases, its toxicity gradually decreases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The single-arm PEG12-modified melittin sequence of the present invention;

[0021] Figure 2 The double-arm PEG12-modified melittin sequence of the present invention;

[0022] Figure 3 The four-arm PEG12-modified melittin sequence of the present invention;

[0023] Figure 4 The present invention verifies the cytotoxicity of melittin modified with different numbers of PEG linking arms; wherein A is the toxicity of unmodified Melittin to cervical cancer cells (Hela); B is the toxicity of Melittin modified with one-arm PEG12 to Hela cells; C is the toxicity of Melittin modified with two-arm PEG12 to Hela cells; and D is the toxicity of Melittin modified with four-arm PEG12 to Hela cells. DETAILED DESCRIPTION

[0024] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0025] Embodiment 1: The present invention provides a method for preparing PEG-modified pharmaceutical polypeptides with different numbers of linker arms, comprising the following steps:

[0026] S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, then removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM, wherein the amount of methylformamide DMF and DCM can fully agitate the resin; diisopropylethylamine DIEA, dimethylformamide DMF, DCM, nitrogen bubbling reaction for 60 minutes; adding methanol, reacting for half an hour, removing the reaction solution, and washing with DMF and MEOH;

[0027] S2, add the second amino acid 2n in the sequence, 1-hydroxyl, benzotriazole tetramethyl hexafluorophosphate HBTU and DIEA to the reactor, and react with N2 bubbling for half an hour, wash off the liquid, and detect with ninhydrin; then cap with pyridine and acetic anhydride; wash, add decapping liquid to remove the 9-fluorenylmethoxycarbonyl Fmoc protecting group, wash, and detect with ninhydrin;

[0028] S3, adding different amino acids in the sequence in sequence and modifying them according to step S2;

[0029] S4. After drying the resin with nitrogen, remove it from the reaction column and pour it into a flask. Then add cutting liquid to the flask. The amount of cutting liquid added is based on the ratio of 10 ml of cutting liquid per gram of resin. The composition of the cutting liquid is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water. Shake and filter out the resin; get the filtrate, add ether to the filtrate, precipitate the crude product, then centrifuge and wash to get the crude product of the sequence; purify the crude product by high performance liquid chromatography, and perform mass spectrometry to determine the successful synthesis of the modified peptide.

[0030] As can be seen from the above, dichlororesin is used as a carrier, a solvent is added to swell, and then the first amino acid in the sequence is added, and then the reagent is added to react. Then, after washing, the amino acids in the sequence are added in sequence and various modifications are performed; finally, cutting is performed to obtain a crude product. The crude product is purified by high performance liquid chromatography, and then mass spectrometry is performed to verify its successful synthesis.

[0031] Example 2: The present invention also provides an activity evaluation of a pharmaceutical polypeptide modified with PEG with different numbers of linkers, wherein the pharmaceutical polypeptide is an anti-tumor polypeptide and an antimicrobial peptide with toxic side effects, including a toxin peptide; different numbers of PEG linkers are modified at the N-terminus of the toxin peptide by solid phase synthesis, and finally different types of chemically modified toxin peptide chains are formed. The number of PEG arms of the polypeptide modified chain is 2-16.

[0032] Examples of the toxin peptide chains used are as follows:

[0033] (1) Unmodified Melittin Sequence:

[0034] GIGAVLKVLTGLPALISWIKR-KRQQ-NH2;

[0035] (2) Single-arm PEG12 modified bee venom peptide sequence Figure 1 shown.

[0036] (3) Double-arm PEG12 modified bee venom peptide sequence Figure 2 shown.

[0037] (4) Four-arm PEG12 modified bee venom peptide sequence Figure 3 shown.

[0038] like Figure 4 As shown, the cytotoxicity of melittin modified with different numbers of PEG linkers was verified. It was found that the N-terminal modified PEG can effectively reduce the toxicity of the toxin peptide to cells, and its toxicity gradually decreases with the increase of the number of PEG linkers.

[0039] From the above, we can see that, taking melittin as a representative of toxin peptides, melittin oligomers can form hydrophilic pores on the cell membrane to interfere with the integrity of the phospholipid bilayer, causing leakage of intracellular substances and changes in osmotic pressure, which in turn leads to the dissolution of cell tissues. PEG modification has a certain effect of reducing the toxicity of toxin peptides, and the toxicity of toxin peptides decreases with the increase in the number of connecting arms of the PEG modified chain.

[0040] The present invention utilizes solid phase synthesis to connect different numbers of PEG linking arms to the hydrophobic end of the toxin peptide, which is simple to operate and easy to prepare; PEG modification can effectively improve the biological stability of the toxin peptide and maintain good dispersibility of the toxin peptide; and PEG modified with different numbers of linking arms can effectively regulate the systemic toxic side effects of the toxin peptide. As the number of PEG linking arms increases, the toxicity of the toxin peptide gradually decreases, which is beneficial to improving the biosafety of the toxin peptide within the effective toxicity range, prolonging the circulation time of the toxin peptide in the body, and reducing its ability to kill normal tissues and cells.

[0041] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in this application. Without departing from the scope of the present invention, other replacements, improvements, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications still falling within the scope of the appended claims.

[0042] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0043] It should be understood that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but for those of ordinary skill having the benefit of this disclosure, without undue experimentation, the development effort will be a routine task of design, fabrication, and production.

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing PEG-modified pharmaceutical polypeptides with different numbers of linker arms, characterized in that: The following steps are involved: S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, then removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM; diisopropylethylamine DIEA, dimethylformamide DMF, DCM, nitrogen bubbling reaction for 60 minutes; adding methanol, reacting for half an hour, removing the reaction solution, and washing with DMF and MEOH; S2, add the second amino acid 2n in the sequence, 1-hydroxyl, benzotriazole tetramethyl hexafluorophosphate HBTU and DIEA to the reactor, and react with N2 bubbling for half an hour, wash off the liquid, and detect with ninhydrin; then cap with pyridine and acetic anhydride; wash, add decapping liquid to remove the 9-fluorenylmethoxycarbonyl Fmoc protecting group, wash, and detect with ninhydrin; S3, adding different amino acids in the sequence in sequence and modifying them according to step S2; S4. After the resin is dried with nitrogen, it is removed from the reaction column and poured into a flask. Then, a cutting solution is added to the flask, shaken, and the resin is filtered out. The filtrate is obtained, and ether is added to the filtrate to precipitate the crude product, which is then centrifuged and washed to obtain the crude product of the sequence. The crude product is purified by high performance liquid chromatography, and the successful synthesis of the modified peptide is determined by mass spectrometry.

2. The method for preparing a PEG-modified pharmaceutical polypeptide with different numbers of linker arms as claimed in claim 1, characterized in that: In the step S1, the first amino acid of 2n in the sequence is added, and when 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM are added, the amounts of dimethylformamide DMF and DCM are such that the resin can be fully agitated.

3. The method for preparing a PEG-modified pharmaceutical polypeptide with different numbers of linker arms as claimed in claim 1, characterized in that: The amount of cutting liquid added in S4 is added according to the ratio of 10 mL / g of resin.

4. The method for preparing a PEG-modified pharmaceutical polypeptide with different numbers of linker arms as claimed in claim 3, characterized in that: The composition of the cutting solution is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water.

5. An activity evaluation of PEG-modified pharmaceutical polypeptides with different numbers of linkers, wherein the modified peptide is prepared according to the preparation method according to any one of claims 1 to 4, characterized in that: The pharmaceutical peptides used are anti-tumor peptides and antimicrobial peptides with toxic side effects, including toxin peptides; different numbers of PEG linker arms are modified at the N-terminus of the toxin peptide by solid phase synthesis, and finally toxin peptide chains with different types of chemical modifications are formed.

6. The activity evaluation of a PEG-modified pharmaceutical polypeptide with different numbers of linker arms as claimed in claim 5, characterized in that: The number of PEG arms of the polypeptide modified chain is 2-16.

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