Preparation method and activity evaluation of connecting arm modified polypeptides with different lengths

Abstract

The invention belongs to the technical field of biomedicine, and provides a preparation method and activity evaluation of connecting arm modified polypeptides with different lengths, and the preparation method comprises the following steps: by taking dichlororesin as a carrier, adding a solvent for swelling and extracting, then adding a first amino acid in a sequence, and then adding a reagent for reaction; cleaning, sequentially adding amino acids in the sequence, and carrying out various modifications; finally, cutting to obtain a crude product; purifying the crude product by high performance liquid chromatography, and then verifying successful synthesis by mass spectrometry; by providing a synthesis method for modifying PEG with different lengths at the N end and the C end of melittin, the defects that existing melittin is poor in biological stability and large in whole-body toxic and side effects are overcome, the in-vivo stability of the melittin is improved, the toxicity of the melittin is regulated and controlled, and the toxicity is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a preparation method of linker-modified polypeptides of different lengths and activity evaluation thereof. Background Art

[0002] As therapeutic drugs for tumors, immunity, and hormone regulation in the body, peptide drugs are widely used in many disease areas, including diabetes, cancer, HIV infection, etc. Peptide drugs have a long history of development. Since the synthesis of the first therapeutic peptide insulin in 1921, more than 100 peptide drugs have been approved worldwide. Therefore, the development of peptide drugs has also become one of the hot spots in pharmaceutical research. Peptide drugs have advantages such as low immunogenicity, low cost and high selectivity, but they also have disadvantages such as weak in vivo stability and short half-life.

[0003] Bee venom is extracted from the venom glands and accessory glands of honey bees, and is a mixture of protein polypeptides, biogenic amines, and non-protein substances. Among them, melittin is an important active ingredient in bee venom. Melittin has potential membrane activity, and its 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. Melittin alone is too toxic to cells and cannot show a good anti-tumor effect within the range of safe doses. At the same time, its stability in the body is very poor, and it is enzymatically hydrolyzed before it can exert its efficacy.

[0004] By modifying melittin, the activity of the peptide can be improved, the pharmacokinetic stability and permeability of the peptide can be enhanced, the half-life of the peptide can be prolonged, and the toxicity can be reduced. There are two main methods for modifying peptides. One is to modify the peptide chain skeleton, and the other is to introduce other groups at the nitrogen and carbon ends for structural optimization and performance modification on the basis of keeping the peptide skeleton unchanged, including polyethylene glycol modification, protein fusion strategy and cholesterol modification.

[0005] Polyethylene glycol (PEG) is a polymer material that is usually soluble in water and a variety of organic solvents and has good stability and compatibility. PEG modification technology is widely used in the chemical modification of a variety of proteins and peptides. PEG modification has the effects of extending half-life, reducing or eliminating immunogenicity, reducing toxic side effects, and enhancing physical, chemical and biological stability.

[0006] Due to the many limitations of existing melittin, such as easy degradation in the body and excessive toxicity, a modification method and activity evaluation strategy of connecting PEG of different lengths to the N-terminus and C-terminus of melittin were proposed, so as to improve the in vivo stability of melittin, regulate its toxicity, reduce systemic toxic side effects, and expand the application range of melittin. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a preparation method of linker-modified polypeptides of different lengths and activity evaluation thereof, aiming to overcome the shortcomings of existing melittin such as poor biological stability and large systemic toxicity, and provide a synthesis method for modifying the N-terminus and C-terminus of melittin with PEG of different lengths, thereby improving the in vivo stability of melittin, and achieving the regulation of melittin toxicity and reducing toxicity.

[0008] According to the first aspect of the present disclosure, a method for preparing linker-modified polypeptides of different lengths is provided, comprising the following steps:

[0009] S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM, diisopropylethylamine, dimethylformamide, dichloromethane, nitrogen bubbling reaction for 60 minutes, then adding methanol, reacting for half an hour, removing the reaction solution, and washing with DMF and MEOH;

[0010] 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, detect with ninhydrin, cap with pyridine and acetic anhydride, wash again, add decapping liquid to remove the 9-fluorenylmethoxycarbonyl Fmoc protecting group, wash, and detect with ninhydrin;

[0011] S3, adding different amino acids in the sequence in sequence and modifying them according to step S2;

[0012] S4. After the resin is dried with nitrogen, it is removed from the reaction column and poured into a flask. A cutting solution is added to the flask, the flask is shaken, and the resin is filtered out. The filtrate is obtained, ether is added to the filtrate to precipitate a crude product, which is then centrifuged and washed to obtain a 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.

[0013] Preferably, the amounts of dimethylformamide (DMF) and dichloromethane (DCM) added in S1 are amounts that can fully agitate the resin.

[0014] Preferably, the amount of the cutting liquid added in S4 is added in a ratio of 10 mL / g to the resin.

[0015] Preferably, the cutting fluid is composed of 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water.

[0016] According to the second aspect of the present disclosure, an activity evaluation of polypeptides modified with linkers of different lengths is proposed, and the anti-tumor polypeptide melittin is used as a model. The melittin is PEGylated at the N-terminus and the C-terminus, and the melittin polypeptide chain is synthesized by a solid phase synthesis method. During the synthesis process, PEG of different lengths is connected to the N-terminus and the C-terminus of melittin to form melittin polypeptide chains with different types of PEG-modified ends.

[0017] Preferably, the PEG repeating unit of the polypeptide modification chain is 4-200.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention synthesizes melittin modified with PEG of different lengths based on peptide solid phase synthesis technology, which is simple to operate and easy to produce. The prepared melittin polypeptide drug is modified with PEG of different lengths at the N / C termini to improve the stability of melittin in vivo. Among them, the N-terminal modified PEG can effectively reduce the toxicity of melittin to cells, and as the length of PEG increases, its toxicity gradually decreases; while the C-terminal modified PEG has almost no effect on the toxicity of melittin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The PEG12 of the present invention modifies the Melittin sequence at the N-terminus;

[0021] Figure 2 The Melittin sequence is modified at the C-terminus by PEG12 of the present invention;

[0022] Figure 3 The present invention verifies the cytotoxicity of different modifications of the NC terminus; wherein A is the toxicity of unmodified Melittin to cervical cancer cells (Hela); B is the toxicity of Melittin modified with PEG12 at the N terminus to Hela cells; and C is the toxicity of Melittin modified with PEG12 at the C terminus to Hela cells. DETAILED DESCRIPTION

[0023] 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.

[0024] Embodiment 1: The present invention provides a method for preparing linker-modified polypeptides of different lengths, comprising the following steps:

[0025] S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM, the amount of dimethylformamide DMF and dichloromethane DCM is the amount that can fully agitate the resin; diisopropylethylamine, dimethylformamide, dichloromethane, nitrogen bubbling reaction for 60 minutes, then adding methanol, reacting for half an hour, removing the reaction solution, and washing with DMF and MEOH;

[0026] 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, detect with ninhydrin, cap with pyridine and acetic anhydride, wash again, add decapping liquid to remove the 9-fluorenylmethoxycarbonyl Fmoc protecting group, wash, and detect with ninhydrin;

[0027] S3, adding different amino acids in the sequence in sequence and modifying them according to step S2;

[0028] S4. After drying the resin with nitrogen, remove it from the reaction column and pour it into a flask. Add cutting fluid to the flask. The amount of cutting fluid is added according to the ratio of 10mL / gram to the resin. The composition of the cutting fluid is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, and 1% water; shake, filter out the resin; obtain the filtrate, add ether to the filtrate, precipitate the crude product, centrifuge, wash, and obtain 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.

[0029] That is, 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 reagents are added for reaction. 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.

[0030] Example 2: The present invention also provides an activity evaluation of peptides modified with connecting arms of different lengths, using the anti-tumor peptide melittin as a model, wherein the melittin is PEGylated at the N-terminus and the C-terminus, and the melittin polypeptide chain is synthesized by a solid phase synthesis method, and during the synthesis process, PEG of different lengths is connected to the N-terminus and the C-terminus of melittin to form melittin polypeptide chains with different types of PEG-modified ends. The PEG repeating unit of the polypeptide modified chain is 4-200.

[0031] The oligomers of melittin can form hydrophilic pores on the cell membrane, interfering with the integrity of the phospholipid bilayer, causing leakage of intracellular substances and causing changes in osmotic pressure, which in turn leads to the dissolution of cell tissues. N-terminal modified melittin has a certain toxicity reduction effect, while C-terminal modification has no toxicity reduction; and the toxicity of melittin decreases with the growth of the N-terminal PEG modified chain.

[0032] The amino acid sequence of the polypeptide used is as follows:

[0033] Unmodified Melittin sequence:

[0034] GIGAVLKVLTGLPALISWIKR-KRQQ-NH2;

[0035] PEG12 modified the Melittin sequence at the N-terminus Figure 1 shown.

[0036] PEG12 modified the Melittin sequence at the C-terminus Figure 2 shown.

[0037] like Figure 3 As shown, PEG of different lengths is connected to the N-terminus and C-terminus of melittin by solid phase synthesis, which is simple to operate and easy to prepare; PEG modification can effectively improve the in vivo stability of the polypeptide and ensure that it is not easy to aggregate in the body; and PEG modified at the ends of peptide chains of different lengths can effectively regulate the toxicity of melittin, among which melittin with PEG modified at the N-terminus can effectively reduce the toxicity of melittin to cells, improve the biosafety of melittin, and reduce its systemic toxic side effects on the body.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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 polypeptides modified with linker arms of different lengths, characterized in that: The following steps are involved: S1, using n dichlororesin as carrier, adding dichloromethane DCM to swell for half an hour, removing DCM, adding the first amino acid of 2n in the sequence, adding 2n diisopropylethylamine DIEA and dimethylformamide DMF and DCM, diisopropylethylamine, dimethylformamide, dichloromethane, nitrogen bubbling reaction for 60 minutes, then 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, detect with ninhydrin, cap with pyridine and acetic anhydride, wash again, 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. A cutting solution is added to the flask, the flask is shaken, and the resin is filtered out. The filtrate is obtained, ether is added to the filtrate to precipitate a crude product, which is then centrifuged and washed to obtain a 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. A method for preparing modified polypeptides with connecting arms of different lengths as claimed in claim 1, characterized in that: The amounts of dimethylformamide (DMF) and dichloromethane (DCM) added in S1 are amounts that can fully agitate the resin.

3. A method for preparing modified polypeptides with different linker lengths according to 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. A method for preparing modified polypeptides with different linker arm lengths 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 linker-modified polypeptides of different lengths, wherein the linker-modified polypeptides of different lengths are prepared according to the preparation method of any one of claims 1 to 4, characterized in that: The anti-tumor polypeptide melittin was used as a model. The melittin was PEGylated at the N-terminus and the C-terminus respectively, and the melittin polypeptide chain was synthesized by a solid phase synthesis method. During the synthesis process, PEG of different lengths was connected to the N-terminus and the C-terminus of melittin to form melittin polypeptide chains with different types of PEG-modified ends.

6. The activity evaluation of a polypeptide modified with a linker arm of different lengths according to claim 5, characterized in that: The PEG repeating unit of the polypeptide modification chain is 4-200.

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