Ion channel based on a poly-polypeptide backbone and uses thereof

Abstract

The application discloses an ion channel based on a poly-polypeptide skeleton and application thereof, and rational use of the poly-polypeptide main chain length difference of different polymerization degrees and the structure-activity relationship difference of different secondary structures of the poly-polypeptide is used in the poly-polypeptide ion channel of the application, the polymerization degree and the secondary structure of the poly-polypeptide are regulated, the length of the poly-polypeptide and the arrangement of the side chain functional groups are regulated, and then the opening or closing of the poly-polypeptide ion channel is regulated, a responsive poly-polypeptide ion channel is designed by introducing a suitable responsive group into the side chain of the poly-polypeptide, and therefore the tumor-specific activation of the poly-polypeptide ion channel is realized, and inspiration is provided for the design and application of the artificial ion channel.

Description

Technical Field

[0001] This invention relates to the field of ion channel preparation technology, and in particular to an ion channel based on a polypeptide backbone and its application. Background Technology

[0002] Transmembrane transport of ions is fundamental to the physiological processes of living organisms, participating in a range of physiological activities such as maintaining osmotic homeostasis, signal transduction, and cell proliferation and apoptosis. Natural ion channels are a class of transmembrane transport proteins containing a rigid, helical ion selector. They achieve highly selective ion capture through coordination with ions via specific amino acid sequences and utilize their helical structure to form ordered nanopores for efficient ion transport. Natural ion channels can respond to external stimuli (such as pH gradients and osmotic pressure changes) by regulating their switching through conformational changes in the selector. However, natural ion channels are structurally complex, volatile, and difficult to purify. Furthermore, the number of channels on the cell membrane is limited, and their function requires specific physiological environments. Therefore, studying the relationship between their structure and properties presents a significant challenge and limits their applications.

[0003] Artificial ion channels are simple synthetic molecular systems that can serve as alternatives to natural ion channels. The transport of ions in artificial ion channels can be divided into two key processes: first, anchoring to the cell membrane through hydrophobic or electrostatic interactions; and then, achieving efficient ion transport through molecular motion, self-assembly, or the orderly arrangement of ion transport units. However, current artificial ion channels are limited in application due to insufficient ion selectivity, lack of or crude gating functions, and poor structural stability.

[0004] The rigidity and helical structure of natural ion channels are important parameters for regulating ion transport. We hypothesize that the rigidity and flexibility of polymer chains are crucial parameters in polymer ion channel design, significantly impacting the arrangement of side chain groups and multivalent interactions within the polymer. On one hand, due to internal rotation of the molecular chain, the side chain groups of flexible polymers are often randomly arranged. In contrast, rigid chains restrict internal rotation, facilitating the ordered arrangement of side chain groups. On the other hand, compared to coiled flexible chains, rigid chains expose more side chain groups and enhance their affinity for other substances. The design of rigid structures contributes to the orderly arrangement of ion transport units and the exposure of membrane anchoring units, thus improving the transport efficiency of polymer ion channels. However, to date, only a few reports on polymer ion channels have been published, primarily focusing on their construction and functional regulation, with little research on the relationship between chain rigidity / flexibility and ion transport efficiency. Meanwhile, although a series of responsive (e.g., UV light or pH) artificial ion channels have been developed, UV light is cytotoxic and has limited penetration depth; and the aforementioned pH-responsive (e.g., pH 4 and pH 10) ion channels cannot respond to the acidic environment of tumor microenvironment (pH 6.8), thus making it difficult to achieve in vivo tumor-targeted delivery and selective activation of tumor cells. Based on the differences in different cellular microenvironments, constructing polymer ion channels for tumor-targeted delivery and specific activation, and elucidating the structure-activity relationship between their chain rigidity / flexibility and ion transport, will broaden the design of biomimetic ion channels and provide new opportunities for their research in disease diagnosis and anti-tumor therapy. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention discloses a polymer ion channel based on a polypeptide backbone. It rationally utilizes the differences in main chain length at different degrees of polymerization and the ordered or disordered arrangement of functional groups in the side chains of different secondary structures. By controlling the degree of polymerization and secondary structure of the polypeptide, the length of the polypeptide and the arrangement of functional groups in the side chains are controlled, thereby regulating the opening or closing of the polypeptide ion channel. By introducing suitable responsive groups into the side chains of the polypeptide, a responsive polypeptide ion channel is designed, thus achieving tumor-specific activation of the polypeptide ion channel. This invention is the first to introduce a polypeptide backbone into the design of artificial ion channels, providing inspiration for the design and application of artificial ion channels.

[0006] The first objective of this invention is to provide an ion channel based on a polypeptide backbone, comprising a polypeptide backbone and polypeptide side chains, wherein the polypeptide side chains contain ion transport groups (R3) and membrane anchoring groups (R4), and the structure of the ion channel is shown in one of formulas (I) or (II):

[0007] ,in:

[0008] The amino acids forming the polypeptide backbone are L-type amino acids, and R3 is... X is a hydrocarbon group with 0-20 carbon atoms, Y is a linking chain, R1 is a substituted or unsubstituted quaternary ammonium group, R2 is a benzocrown ether, and R4 is a membrane anchoring structure (different compounds can be selected to prepare polypeptides modified with different membrane anchoring groups).

[0009] n represents the degree of polymerization of the polypeptide, preferably an integer from 40 to 1000, and x represents the grafting rate, which is 0.01 to 0.99.

[0010] Furthermore, R1 is R5, R6 and R7 are independently selected from hydrogen or alkyl groups, such as methyl, ethyl, propyl and butyl.

[0011] Furthermore, the crown ether in R2 is selected from 18-crown-6 ether, 15-crown-5 ether, 21-crown-7 ether, or 24-crown-8 ether.

[0012] Furthermore, R3 is , where a is an integer from 0 to 20, b is an integer from 0 to 20, and R5 and R6 are independently selected from hydrogen or alkyl groups. For example The types of crown ethers can be replaced as needed.

[0013] Furthermore, R4 is selected from , , or .

[0014] Polypeptides are protein analogs with good biocompatibility, biodegradability, and ordered secondary structures, including α-helices, β-sheets, and random coils. Compared with other polymers, they possess unique properties, thus having wide applications in drug delivery, antibacterial applications, and antitumor immunotherapy. This invention demonstrates through molecular dynamics simulations that polypeptides first contact the negatively charged cell membrane through cationic groups, then anchor themselves to the cell membrane using the rigid structure of the main chain and the hydrophobic interactions of the side chains, and finally penetrate the cell membrane through the cationic groups of the side chains. This invention, through research on the properties of the polypeptide secondary structure (such as flexibility and length), reveals that under specific conditions (such as degree of polymerization and configuration), polypeptides can function as ion channels with high transport efficiency.

[0015] Furthermore, the structure of the polypeptide is one of the following:

[0016] .

[0017] In this invention, the polypeptide backbone can be derived from a natural amino acid sequence or from an artificially synthesized amino acid sequence. Typically, the polypeptide backbone comprises 40-1000 amino acid residues, preferably 40-500 amino acid residues, and more preferably 40-200 amino acid residues.

[0018] Furthermore, the amino acids are naturally occurring amino acids and their derivatives. Naturally occurring amino acids include, but are not limited to, glutamic acid, tyrosine, serine, homoserine, lysine, cysteine, histidine, valine, arginine, glutamine, glycine, leucine, tryptophan, etc., and their derivatives include, but are not limited to, glutamate esters, serine esters, and homocysteine, etc.

[0019] Furthermore, the polypeptide backbone may include residues of one or more amino acids or their derivatives. For example, it may include residues of two amino acids or their derivatives, or residues of three or four amino acids or their derivatives.

[0020] Furthermore, the polypeptide backbone may be poly(γ-propyne- L β-Glutamate benzyl ester, poly( L -Tyrosine), Poly( L -Lysine), Poly( L -serine), poly( L -high serine), poly( L -cysteine, poly( L -Glutamic acid-γ-propargyl ester, poly(γ-3-chloropropyl- L -Glutamate) or poly(γ-3-chlorohexyl- L (e.g., glutamate).

[0021] A second objective of this invention is to provide a method for preparing the aforementioned polypeptide ion channel, comprising the following steps:

[0022] Using amine, transition metal initiator, base or amino-modified nanoparticles as initiators, two or more compounds of Formula III (N-carboxylic anhydride monomers (NCA) of amino acids or their derivatives) are prepared by ring-opening polymerization to obtain a polypeptide backbone. A membrane anchoring group is grafted onto the polypeptide side by click chemistry (grafting R4), and an ion transport group is grafted onto the polypeptide side by click chemistry and quaternization reaction (grafting R3) to obtain the polypeptide ion channel.

[0023] ;

[0024] Where A is an amino acid or its derivative.

[0025] Furthermore, N,N-dimethylformamide, dichloromethane, or a mixture of dichloromethane and water are used as solvents.

[0026] Furthermore, in the initiator, the amine can be n-butylamine, n-hexylamine, propargylamine, triethylamine, and hexamethylenediamine, etc.; the transition metal initiator can be organonickel or organocobalt, etc.; and the base can be hexamethyldisilazane, hexamethyldisilazane lithium nitride, and sodium bicarbonate, etc.

[0027] Furthermore, the click chemistry reaction is specifically a Cu(I)-catalyzed azido-alkynyl 1,3-dipolar cycloaddition reaction. Generally, the reaction is carried out at room temperature (20-25 °C) for 24-36 hours, with azide and alkynyl as active groups, cuprous bromide as catalyst, pentamethyldiethylenetriamine as complexing agent, and azido-alkynyl as active group.

[0028] Furthermore, the quaternization reaction is specifically a reaction between a tertiary amine group and a haloalkanes. Generally, N,N-dimethylformamide and acetonitrile are used as solvents, and sodium iodide is added to the system as a catalyst. The reaction is carried out at 60-100 °C for 1-90 hours. After the reaction is completed, a saturated NaCl solution is added, and the product is dialyzed in the NaCl solution for ion exchange.

[0029] In one embodiment of the present invention, the polymerized side-functionalized polypeptide backbone, azid tertiary amine molecule, and azid cholesterol are dissolved in N,N-dimethylformamide, and a membrane anchoring group is introduced in the presence of pentamethyldiethylenetriamine and cuprous bromide. Subsequently, the product with the introduced membrane anchoring group is reacted in the presence of sodium iodide and haloalkanes to obtain a polypeptide with side chains connected to membrane anchoring groups and ion transport groups, respectively.

[0030] A third objective of this invention is to provide a polypeptide-based responsive ion channel comprising a polypeptide backbone and polypeptide side chains, wherein the polypeptide side chains contain a membrane anchoring group (R4), an ion transporting group (R3), a cation group, and an anion group, and the structure of the responsive ion channel is shown in one of formulas (IV)-(VII):

[0031] ,in:

[0032] The amino acids forming the polypeptide backbone are L-type amino acids, and R3 is... X is a hydrocarbon group with 0-20 carbon atoms, Y is a linking chain, R1 is a substituted or unsubstituted quaternary ammonium salt group, R2 is a benzocrown ether, R4 is a membrane anchoring structure, and R8 is an anionic group.

[0033] n+y represents the degree of polymerization of the polypeptide, where n+y is an integer from 40 to 1000, x is from 0.01 to 0.99, and n / y ≥ 4. The preferred constraints for R1-R4 are the same as above.

[0034] Furthermore, R8 includes a carboxyl group or a phosphate group.

[0035] Furthermore, R8 is selected from , or .

[0036] In this invention, the introduction of anionic groups prevents the polymer from functioning as an ion channel before reaching the target site and responding. After the target site responds, the anionic groups detach, leaving only cationic groups, which then function. Furthermore, current ion channels mainly include small molecule ion channels and self-assembled supramolecular ion channels. Monomolecular ion channels or transporters require precise control of molecular chain length to match cell membrane thickness, thus presenting certain challenges in synthesis. Self-assembled supramolecular ion channels are unstable, and their assembly structure and transport efficiency are easily affected by changes in the cellular environment. The polypeptide ion channel of this invention effectively solves these problems.

[0037] Furthermore, the structure of the responsive polypeptide ion channel is shown in one of the following examples:

[0038] .

[0039] The responsive ion channels described in this invention possess pH-responsive, phosphatase-responsive, and reactive oxygen species (ROS)-responsive properties, enabling conformational changes and ion channel activation triggered by pH, phosphatase, or ROS concentration variations. Specifically, co-incubating responsive polypeptide ion channels with carboxyl anionic groups with a slightly acidic environment significantly enhances ion transport efficiency compared to unincubated conditions. Similarly, co-incubating responsive polypeptide ion channels with phosphate anionic groups with phosphatase or hydrogen peroxide significantly enhances ion transport efficiency compared to unincubated conditions. Among these, responsive polypeptide ion channels with aconitine anionic groups are pH-responsive.

[0040] Furthermore, this application primarily leverages the pH difference between normal physiological conditions and the tumor microenvironment to achieve the switching function of polypeptide ion channels. Under normal physiological conditions, responsive polypeptides are flexible, negatively charged random coil structures. At this time, due to the electrostatic attraction between the side chains of the polypeptide and the disordered arrangement of ion transport groups, they lack ion transport capacity. After microacid treatment, the carboxylate groups on the side chains are removed, and the polypeptide transforms into a rigid, positively charged α-helix structure. The ion transport groups on the side chains are arranged in an orderly manner, giving it ion transport capacity, thus realizing the process of the polypeptide ion channel changing from "closed" to "open".

[0041] Furthermore, phosphatase-responsive polypeptide ion channels can be prepared by replacing lysine with terminal tyrosine in one embodiment of the present invention, and then grafting phosphate groups with phosphatase responsiveness onto the tyrosine portion. Under normal physiological conditions, polypeptides are flexible, negatively charged random coil structures. At this time, due to the electrostatic attraction between the side chains of the polypeptide, the disordered arrangement of ion transport groups results in a lack of ion transport capacity. After phosphatase treatment, the phosphate groups on the side chains are removed, and the polypeptide transforms into a rigid, positively charged α-helix structure. The ion transport groups on the side chains are arranged in an orderly manner, giving it ion transport capacity, thus realizing the process of the polypeptide ion channel changing from "closed" to "open".

[0042] Furthermore, ROS-responsive polypeptide ion channels can be prepared by replacing lysine with terminal serine or homoserine in one embodiment of the present invention, and then introducing a carboxyl group with ROS responsiveness as shown in the following formula at the end of the serine or homoserine. Under normal physiological conditions, polypeptides are flexible, negatively charged random coil structures. At this time, due to the electrostatic attraction between the side chains of the polypeptide, the disordered arrangement of ion transport groups results in a lack of ion transport capacity. After hydrogen peroxide treatment, the carboxyl group on the side chain is removed, and the polypeptide transforms into a rigid, positively charged α-helix structure. The side chain ion transport groups are arranged in an orderly manner, giving it ion transport capacity, thus realizing the process of the polypeptide ion channel changing from "closed" to "open". The structure of the terminal serine with a hydroxyl group is as follows:

[0043] .

[0044] Furthermore, the aforementioned ion channels are either exogenous or endogenous ion channels.

[0045] A fourth objective of this invention is to provide a method for preparing the above-mentioned responsive polypeptide ion channel, comprising the following steps:

[0046] S1. Using n-butylamine as an initiator, two or more compounds shown in Formula III (N-carboxylic anhydride monomers (NCA) of amino acids or their derivatives) are randomly copolymerized to obtain a polypeptide backbone.

[0047] S2. By linking membrane anchoring groups to the polypeptide side through click chemistry, by linking cationic groups and ion transport groups to the polypeptide side through click chemistry and quaternization, and by linking anionic groups to the polypeptide side through amidation, phosphorylation or esterification, the responsive polypeptide ion channel is obtained.

[0048] ;

[0049] Where A is an amino acid or its derivative.

[0050] Furthermore, in random copolymerization, the solvent is N,N-dimethylformamide, dichloromethane, or a mixture of dichloromethane and water.

[0051] A fifth objective of this invention is to provide the application of the above-mentioned polypeptide ion channels or responsive polypeptide ion channels in the preparation of ion transport products or drug delivery products (especially pharmaceuticals).

[0052] A sixth objective of this invention is to provide the application of the above-mentioned polypeptide ion channels or responsive polypeptide ion channels in the preparation of diagnostic or therapeutic products.

[0053] Furthermore, the aforementioned polypeptide ion channels or responsive polypeptide ion channels can be used in the preparation of diagnostic or therapeutic drugs for tumors or ion channelopathies, such as antitumor drugs, therapeutic drugs for diseases with defects in natural ion channels (many diseases (such as cystic fibrosis, certain arrhythmias, and neurological diseases) originate from functional defects in natural ion channels. Artificial ion channels may serve as alternatives or adjunct devices to restore normal ion balance in cells, providing new strategies for treating these diseases), etc. They can also be used as drug delivery platforms and tumor-killing drugs.

[0054] Furthermore, the above-mentioned polypeptide ion channels or responsive polypeptide ion channels can be used in the preparation of diagnostic or therapeutic drugs for inflammation or infection, such as anti-inflammatory agents and antibacterial agents. Since sites of inflammation or bacterial infection have a slightly acidic environment and / or high ROS concentrations, the ion channels of this invention can be used for the diagnosis and treatment of inflammation or infection.

[0055] By means of the above-described solution, the present invention has at least the following advantages:

[0056] This invention utilizes the difference in compliance between different conformations of polypeptides to design a conformationally tunable polypeptide. This polypeptide can regulate the ordered or disordered arrangement of functional groups on the side chains of polypeptide ion channels, thereby controlling the "on" or "off" of polypeptide ion channels. This cleverly simulates the allosteric process of natural ion channel proteins in the synthetic system, providing guidance for the study of structure-activity relationships of polypeptides, the design of artificial ion channels, and the design of smart nanomaterials. Attached Figure Description

[0057] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0058] Figure 1 The 1H NMR spectrum of azide cholesterol in Example 1;

[0059] Figure 2 The 1H NMR spectrum of CE-Br in Example 2;

[0060] Figure 3 The 1H NMR spectrum of PPOBLG (DP = 60) in Example 4;

[0061] Figure 4 The hydrogen NMR spectrum of PPOBDLG (DP = 60) in Example 4;

[0062] Figure 5 The gel permeation chromatograms of PPOBLG (DP = 60) and PPOBDLG (DP = 60) in Example 4 are shown.

[0063] Figure 6 The image shows the proton NMR spectrum of the PCA (DP = 60) in Example 5.

[0064] Figure 7 The 1H NMR spectrum of PCAE (DP = 60) in Example 6;

[0065] Figure 8 The circular dichroism chromatogram of PCAE (DP = 60) in Example 6;

[0066] Figure 9 PAEK in Example 9 Boc The proton NMR spectrum;

[0067] Figure 10 PAEK in Example 9 Boc Gel permeation chromatogram;

[0068] Figure 11 PCAK in Example 10 Boc The proton NMR spectrum;

[0069] Figure 12 The diagram shows the potassium ion transport capacity of PCAE(L) and PCAE(DL) in test example (1) and the potassium / sodium ratio of PCAE(L).

[0070] Figure 13 For test case (1) PCAEK CA and PCAEK SADiagram of potassium ion transport capacity under different pH conditions;

[0071] Figure 14 The membrane anchoring capability diagram of PCAE(L, DP = 60) and PCAE(DL, DP = 60) verified by the FRET effect in test example (2);

[0072] Figure 15 Cell membrane anchoring diagrams of PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) in test example (3);

[0073] Figure 16 The intracellular potassium ion efflux map induced by PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) in test example (4);

[0074] Figure 17 For test case (4) PCAEK CA and PCAEK SA Maps of intracellular potassium ion efflux induced under different pH conditions;

[0075] Figure 18 The graph shows the intracellular reactive oxygen species induced by PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) in test example (5).

[0076] Figure 19 For test case (5) PCAEK CA and PCAEK SA Map of intracellular reactive oxygen species induced under different pH conditions;

[0077] Figure 20 The polarization of mitochondrial membrane potential induced by PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) in test example (6) is shown.

[0078] Figure 21 For test case (6) PCAEK CA and PCAEK SA Mitochondrial membrane potential polarization induced under different pH conditions;

[0079] Figure 22 The diagram shows the cytochrome C release induced by PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) in test example (7);

[0080] Figure 23 For test case (7) PCAEK CA and PCAEK SA Graphs showing the induction of cytochrome C release under different pH conditions;

[0081] Figure 24 Flow cytometry and quantitative data of apoptosis induced by PCAE(L, DP = 10, 20, 40, 60, 80) and PCAE(DL, DP = 10,20, 40, 60, 80) in test case (8);

[0082] Figure 25 For test case (8) PCAEK CA and PCAEK SA Flow cytometry and quantitative data of apoptosis induced under different pH conditions;

[0083] Figure 26 The graph shows the cytotoxicity of PCAE(L, DP = 10, 20, 40, 60, 80) and PCAE(DL, DP = 10,20, 40, 60, 80) at different concentration gradients in test example (9).

[0084] Figure 27 For test case (9) PCAEK CA and PCAEK SA Cytotoxicity test results under different pH conditions;

[0085] Figure 28 Tumor volume, survival rate, HE staining and TUNEL staining sections of tumor-bearing mice after administration in test case (10);

[0086] Figure 29 The graph shows the weight changes of tumor-bearing mice after drug administration in test case (10);

[0087] Figure 30 The H&E stained sections of the major organs after drug administration in test case (10) are shown. Detailed Implementation

[0088] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0089] In the embodiments, L-Glutamic acid, n-butylamine, triethylamine, pentamethyldiethylenetriamine, cuprous bromide, cholesterol bromide, carboxybenzo-18-crown-6 ether, etc., were purchased from Aladdin Chemical; 4-dimethylaminopyridine, triphosgene, cis-aconitine anhydride, succinic anhydride, potassium azide, iodomethane, sodium iodide, phosphoryl chloride, and 1H-pyrazole-1-formamidinium hydrochloride were purchased from Anaiji Chemical; γ-(4-propoxybenzyl)- L -Glutamic acid-N-carboxylic anhydride monomer (POBLG-NCA) was synthesized according to the method described in previous literature (J AmChem Soc, 2023, 145, 11206-11214); water-soluble Cy5 active ester (Cy5-NHS), water-soluble Cy3 active ester (Cy3-NHS), and mitochondrial membrane potential detection kit (JC-1) were purchased from Meilun Biotechnology; potassium ion probes EPG-4 AM and Pluronic F-127 were purchased from Maokang Biotechnology (Shanghai, China). Hydrogen peroxide detection kit and apoptosis detection kit were purchased from Beyotime (Shanghai, China). Cytochrome C antibody was purchased from Abclonal (Wuhan, China). All glassware used was purchased from Xinweier.

[0090] Analytical balance purchased from Sartorius (model: BSA224S). Magnetic stirrer purchased from IKA (model: RHdigital). Centrifuge purchased from Thermo SCIENTIFIC (model: MULTIFUGE X1R). Rotary evaporator purchased from IKA (model: RV10). Circular dichroism chromatograph (CD, model: J-900). Fluorescence spectrometer (FLUOROMAX-4). Gel permeation chromatograph (GPC, 1260 Infinity). Laser confocal microscope (CLSM, ZEISS 800). 1H NMR spectrometer (…). 1 H NMR (400 MHz). Multifunctional microplate reader (A-2082). Flow cytometer (Fongcyte).

[0091] Unless otherwise specified, percentages (%) refer to molar percentages relative to the composition; the components involved or their preferred components can be combined to form new technical solutions; all mentioned embodiments and preferred embodiments can be combined to form new technical solutions; all mentioned technical features and preferred features can be combined to form new technical solutions; the sum of the contents of all components in the composition is 100%; the sum of the parts of all components in the composition can be 100 molar parts; the numerical range represents an abbreviation of any combination of real numbers from a to b, where a and b are real numbers. For example, the numerical range "0-20" means that all real numbers between "0-20" have been listed in this document, and "0-20" is just an abbreviation of these numerical combinations; the integer numerical range "ab" represents an abbreviation of any combination of integers from a to b, where a and b are integers. For example, the integer range “1-N” means 1, 2...N, where N is an integer; “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture; the term “a” means “at least one”; the percentages (including molar percentages) are based on the total amount of substance of the composition.

[0092] The term "range" as disclosed herein takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable, meaning that any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–80 is listed for a specific parameter, it is expected that a range of 10–300 is also expected. Furthermore, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5; the term "residue" refers to the corresponding portion in the product (e.g., a polymer) formed after a reaction of a compound or monomer; "amino acid" generally includes amino acids and their derivatives.

[0093] Methods for forming α-helical polypeptide backbones from amino acids and regulating their secondary structures are known in the art. For example, see J Am Chem Soc, 2023, 145, 11206-11214; Chem Soc Rev, 2018, 47:7401–7425; J Mater Chem B, 2020, 8: 6530–6547; Angew Chem Int Ed, 2017, 56:10826–10829; Proc Natl Acad Sci USA, 2017, 114: 12675–12680. Specifically, using n-butylamine as an initiator and N,N-dimethylformamide as a solvent, ring-opening polymerization is used to initiate random copolymerization of N-carboxylic anhydride monomers (NCA) based on one or two amino acids and their derivatives. Branch membrane anchoring groups (such as cholesterol), cationic quaternary ammonium salt groups, ion transport groups (such as crown ethers), responsive anionic carboxylate or phosphate groups are linked to the polypeptide side to prepare a polypeptide with adjustable secondary structure. The secondary structure can be adjusted by pH, phosphatase or ROS response.

[0094] In the following embodiments, the polypeptide is referred to as PCAE(L), PCAE(DL), or PCAEK, depending on the type and conformation of its side groups. CA PCAEK SA PCAEB C PCAEB P Where P represents the polypeptide backbone; C represents cholesterol; A represents a quaternary ammonium group; E represents 18-crown-6 ether; K represents lysine; B represents serine, CA Represents cis-aconitine. SA Represents succinic anhydride; C Represents ROS-responsive groups; P represents the phosphatase-responsive group; n represents the degree of polymerization of the polypeptide; x represents the grafting rate of ion transport units in the polypeptide.

[0095] The following reaction procedures illustrate part of the preparation of the polypeptide ion channels described in this application:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Where n = 10, 20, 40, 60 or 80; x = 0.80.

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Where n + y = 10, 20, 40, 60 or 80, n / y = 4 / 1; x = 0.80.

[0110] Preferably, in the above process, the degree of polymerization of the polypeptide ion channel is 40, 60 or 80, the cation used is a quaternary ammonium salt, the membrane anchoring group used is cholesterol, the ion transport unit used is 18-crown-6, and the anionic group used is a carboxyl group.

[0111] The polypeptide described in this application can regulate the arrangement of the side chain groups of the polypeptide through conformational changes (including ordered and disordered arrangement; of course, the polypeptide ion channel of this invention is only one application form of the polypeptide of this invention, and its polypeptide side chains can link different functional groups to achieve applications in different fields), thereby controlling the opening or closing of the polypeptide ion channel.

[0112] Example 1: Preparation of azide cholesterol

[0113] Brominolide (0.9 g, 2 mmol) and potassium azide (0.49 g, 6 mmol) were dissolved in N,N-dimethylformamide (10 mL) and reacted at 92 °C for 48 hours. After the reaction was complete, the solution was added dropwise to deionized water (100 mL) and extracted with dichloromethane (2 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to obtain brominolide. Its 1H NMR spectrum is shown below. Figure 1 .

[0114]

[0115] Example 2 Preparation of 4-(6-bromohexyl)-carboxybenzo18-crown 6 ether

[0116] 6-Bromo-1-hexanol (1.02 g, 5.63 mmol), 4-carboxybenzo-18-crown-6 ether (1 g, 2.81 mmol), and 4-dimethylaminopyridine (172 mg, 1.41 mmol) were dissolved in dichloromethane (70 mL) and stirred at 0 °C for 10 min. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (0.54 g, 2.81 mmol) was added to the reaction mixture, and the reaction was continued at 40 °C for 60 h. After the reaction was complete, the mixture was centrifuged (10,000 rpm, 10 min), and the supernatant was collected. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography, using dichloromethane and dichloromethane / methanol (v / v = 80 / 20) as eluents, respectively. Finally, the product was dissolved in dichloromethane (50 mL), washed with hydrochloric acid (2 × 30 mL, 1 mol / L), and then washed with saturated sodium bicarbonate solution (1 × 30 mL). The organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to give a white solid 4-(6-bromohexyl)-carboxybenzo-18-crown-6 ether. Its 1H NMR spectrum is shown below. Figure 2 .

[0117]

[0118] Example 3 Preparation of N,N-dimethylazidopropylamine

[0119] Dissolve N,N-dimethylchloropropylamine hydrochloride (1.0 g, 6.33 mmol) in deionized water (10 mL), add sodium azide (0.82 g, 12.65 mmol), 70 o The reaction was carried out at C for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, the pH was adjusted to 12 with sodium hydroxide solution (0.1 mol / L), and extracted with anhydrous diethyl ether (30 mL × 5). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to obtain N,N-dimethylazidopropylamine.

[0120]

[0121] Example 4: Preparation of PPOBLG and PPOBDLG with different degrees of polymerization

[0122] Polypeptides PPOBLG and PPOBDLG with different degrees of polymerization initiated by n-butylamine were synthesized by adjusting the degree of polymerization. Taking PPOBLG with a degree of polymerization of 60 as an example, POBLG-NCA (100 mg, 0.315 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL) in a glove box, and a solution of n-butylamine in N,N-dimethylformamide (53 µL, 0.1 mol / L, [M]0 / [I]0 = 60) was added. The reaction was carried out at room temperature for 72 hours. The reaction progress was monitored by Fourier transform infrared spectroscopy until the monomer conversion rate reached 100%. After the reaction was completed, the solution was added dropwise to deionized water (100 mL) with vigorous stirring to obtain a white solid. The solution was washed three times with deionized water to remove residual impurities and dried under vacuum to obtain white solid PPOBLG.

[0123] PPOBLG of other degrees of polymerization were synthesized using the same steps described above. PPOBDLG of different degrees of polymerization were synthesized from POBDLG-NCA using the same steps described above.

[0124] Fluorescently labeled polypeptides were prepared by labeling the main chain ends with fluorescent groups. After the monomer conversion reached 100%, a dimethyl sulfoxide solution of Cy3-NHS or Cy5-NHS (5 mg / mL, 50 µL) and N,N-diisopropylethylamine (10 µL) were added, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the solution was added dropwise to deionized water (60 mL) with vigorous stirring to obtain... Cy3 PPOBLG, Cy5 PPOBLG, Cy3 PPOBDLG, Cy5 PPOBDLG. Washed three times with deionized water to remove residual impurities, then lyophilized to obtain blue (Cy5 labeled) and pink (Cy3 labeled) solids. Their proton NMR spectra are shown below. Figure 3 and Figure 4 The resulting solid was dissolved in N,N-dimethylformamide containing LiBr (0.05 mol / L) to achieve a polymer concentration of 5 mg / mL. The solution was filtered through a 0.22 µm organic filter, and the molecular weight and polydispersity of the polypeptide were determined by GPC. The GPC chromatogram and data are shown below. Figure 5 .

[0125]

[0126] Example 5 Synthesis of Polypeptide PCA via Click Chemistry

[0127] Taking PCA(L) with a degree of polymerization of 60 as an example. In a glove box, PPOBLG (30 mg, 0.11 mmol of alkynyl group), N,N-dimethyl-3-azidopropylamine (12.4 mg, 0.097 mmol), and azidocholesterol (10 mg, 0.024 mmol) were dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of pentamethyldiethylenetriamine (34 µL, 0.125 mmol) and cuprous bromide (18 mg, 0.125 mmol). The reaction was carried out at room temperature for 48 hours. After the reaction was completed, the mixture was exposed to air to oxidize the excess cuprous bromide, and hydrochloric acid (3-4 mL, 1 mol / L) was added until the solution changed from blue to yellow. The crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), lyophilized, and a white flocculent solid PCA(L) was obtained. Its 1H NMR spectrum is shown below. Figure 6 .

[0128]

[0129] Example 6 Synthesis of polypeptide PCAE via quaternary ammonium salt reaction

[0130] Taking PCAE(L) with a degree of polymerization of 60 as an example, PCA(L) (37 mg, 0.09 mmol of tertiary amine group) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and sodium iodide (124 mg, 0.83 mmol) and CE-Br (143 mg, 0.28 mmol) were dissolved in acetonitrile (2 mL). The two mixtures were then reacted at 80 °C for 48 hours. After the reaction, saturated sodium chloride solution (4 mL) was added to the mixture, and the mixture was stirred at room temperature for 4 hours to perform ion exchange. After the reaction, the crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), then lyophilized to obtain a white solid, PCAE(L). Its 1H NMR spectrum is shown below. Figure 7 .

[0131]

[0132] Characterizing the conformation of PCAE using CD, such as Figure 8 As shown, PCAE(L) is a rigid α-helical conformation, and the helicity increases with the degree of polymerization, while PCAE(DL) is a random coil conformation.

[0133] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0134]

[0135] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0136]

[0137] Example 7

[0138] γ-propynyl- L 1.0 g (5.4 mmol) of glutamate and 0.8 g (2.7 mmol) of triphosgene were dissolved in anhydrous tetrahydrofuran (25 mL), and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, the tetrahydrofuran and residual triphosgene were removed under vacuum to give a pale yellow liquid. The crude product was then dissolved in ethyl acetate (50 mL), washed with saturated sodium bicarbonate (50 mL × 3) and saturated sodium chloride (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum to give a colorless viscous liquid, PLG-NCA.

[0139]

[0140] Taking the synthesis of PPLG-2 with a degree of polymerization of 60 as an example, POBLG-NCA was replaced with PLG-NCA, and the n-butylamine-induced polypeptide PPLG-2 was synthesized according to the method in Example 4.

[0141]

[0142] Based on PPLG-2, PCA-2 was synthesized by click chemistry according to the method in Example 5.

[0143]

[0144] Based on PCA-2, PCAE-2 was synthesized by quaternization reaction according to the method in Example 6.

[0145]

[0146] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0147]

[0148] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0149]

[0150] Example 8

[0151] N(e)-tert-butoxycarbonyl- L -Lysine (1.1 g, 4.4 mmol) and triphosgene (0.58 g, 1.9 mmol) were dissolved in anhydrous tetrahydrofuran (25 mL), 50 o The reaction was carried out at C for 1 hour. After the reaction was completed, the insoluble matter was removed by filtration, and tetrahydrofuran and residual triphosgene were removed under vacuum to obtain a white solid. Then, tetrahydrofuran (10 mL) was added to dissolve the crude product, and n-hexane (100 mL) was added to precipitate the product. This process was repeated three times to obtain Boc-Lys-NCA.

[0152]

[0153] Example 9 PAEK Boc Preparation

[0154] PAEK was prepared by random copolymerization of POBLG-NCA and Boc-lys-NCA initiated by n-butylamine. Boc POBLG-NCA (900 mg, 2.84 mmol) and Boc-lys-NCA (193 mg, 0.71 mmol) were dissolved in anhydrous N,N-dimethylformamide (6 mL), followed by the addition of a solution of n-butylamine in N,N-dimethylformamide (591 µL, 0.1 mol / L, [M]0 / [I]0 = 60). The reaction was carried out at room temperature for 72 hours. The reaction progress was monitored by Fourier transform infrared spectroscopy until the monomer conversion reached 100%. After the reaction was completed, the solution was added dropwise to deionized water (100 mL) under vigorous stirring to obtain a white solid. The crude product was washed three times with deionized water and dried under vacuum to obtain a white solid, PAEK. Boc Its proton NMR spectrum is shown below. Figure 9 .

[0155] The resulting solid was dissolved in N,N-dimethylformamide containing LiBr (0.05 mol / L) to achieve a polymer concentration of 5 mg / mL. The solution was filtered through a 0.22 µm organic filter, and the molecular weight and polydispersity of the polypeptide were determined by GPC. The GPC chromatogram and data are shown below. Figure 10 .

[0156]

[0157] Example 10 Synthesis of polypeptide PCAK via click chemistry Boc

[0158] In the glove box, place PAEK BocN,N-dimethyl-3-azidopropylamine (30 mg, 0.09 mmol), N,N-dimethyl-3-azidopropylamine (10.1 mg, 0.079 mmol), and azidocholesterol (8.2 mg, 0.02 mmol) were dissolved in anhydrous N,N-dimethylformamide, followed by the addition of pentamethyldiethylenetriamine (34 μL) and cuprous bromide (18 mg). The reaction was carried out at room temperature for 36 hours. After the reaction was completed, the mixture was exposed to fish air to oxidize the excess cuprous bromide, and hydrochloric acid (3-4 mL, 1 mol / L) was added until the solution changed from blue to yellow. The crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), lyophilized, and given as a white solid PCAK. Boc Its proton NMR spectrum is shown below. Figure 11 .

[0159]

[0160] Example 11 Preparation of PCAK via Boc De-oxidation

[0161] PCAK Boc (500 mg, with a molecular weight of 0.26 mmol of Boc group on the side chain of the polypeptide) was dissolved in trifluoroacetic acid / dichloromethane (5 mL, v / v = 1 / 3) and reacted at room temperature for 3 hours. After the reaction was complete, the solvent was removed under vacuum to obtain a crude product as a yellow solid. To further remove trifluoroacetic acid, the crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), then lyophilized to obtain a white solid, PCAK.

[0162]

[0163] Example 12 Preparation of PCAEK via Quaternization Reaction

[0164] PCAK (46 mg, 0.09 mmol of tertiary amine group) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and sodium iodide (124 mg, 0.83 mmol) and CE-Br (143 mg, 0.28 mmol) were dissolved in acetonitrile (2 mL). These two mixtures were then reacted at 80 °C for 48 hours. After the reaction, saturated sodium chloride solution (4 mL) was added to the system, and the mixture was stirred at room temperature for 4 hours to perform ion exchange. After the reaction, the crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), and then lyophilized to obtain a white solid, PCAEK.

[0165]

[0166] Example 13 Preparation of PCAEK CA and PCAEK SA

[0167] PCAEK (30 mg, with a primary amine group of 0.016 mmol) and cis-aconitine anhydride (17 mg, 0.105 mmol) were dissolved in deionized water (2 mL). Sodium hydroxide solution (0.2 mol / L) was added to maintain the pH at approximately 9, and the reaction was carried out at room temperature for 12 hours. After the reaction was complete, the crude product was dialyzed against slightly alkaline (pH 9-10) deionized water for one day (MWCO = 3500 Da), and then lyophilized to obtain a white solid PCAEK. CA .

[0168]

[0169] PCAEK (30 mg, with a primary amine group of 0.016 mmol on the side chain) and succinic anhydride (11 mg, 0.105 mmol) were dissolved in deionized water (2 mL). Sodium hydroxide solution (0.2 mol / L) was added to maintain the pH at approximately 9, and the reaction was carried out at room temperature for 12 hours. After the reaction was complete, the crude product was dialyzed against slightly alkaline (pH 9-10) deionized water for one day (MWCO = 3500 Da), and then lyophilized to obtain a white solid PCAEK. SA .

[0170]

[0171] Example 14 Synthesis of phosphatase-responsive polypeptide ion channels

[0172] O-tert-butyl- L β-serine (1.0 g, 6.2 mmol) and triphosgene (1.2 g, 4.13 mmol) were dissolved in anhydrous tetrahydrofuran (25 mL), 50 o The reaction was carried out at C for 3 hours. After the reaction was completed, tetrahydrofuran and residual triphosgene were removed under vacuum to obtain a white solid. Then, tetrahydrofuran (5 mL) was added to dissolve the crude product, and n-hexane (60 mL) was added to precipitate the product. This process was repeated three times to obtain Ser(tBu)-NCA.

[0173]

[0174] Replace Boc-Lys-NCA with Ser(tBu)-NCA and synthesize PAEB(tBu) according to the method in Example 9.

[0175]

[0176] Based on PAEB(tBu), PCAB(tBu) was synthesized by click chemistry according to the method in Example 10.

[0177]

[0178] Based on PCAB(tBu), PCAB was synthesized by deprotection reaction according to the method in Example 11.

[0179]

[0180] Based on PCAB, PCAEB was synthesized by quaternization reaction according to the method in Example 12.

[0181]

[0182] PCAEB was synthesized via phosphorylation. P PCAEB (30 mg) was dispersed in anhydrous N-methylpyrrolidone (3 mL), and anhydrous triethylamine (200 μL) and phosphoryl chloride (200 μL) were slowly added dropwise under ice bath conditions. The reaction was carried out in the dark at room temperature for 12 hours. After the reaction was completed, saturated sodium bicarbonate solution (7-8 mL) was added to the reaction solution, and the reaction was continued at room temperature for 2 hours. The crude product was dialyzed against deionized water for three days (MWCO = 3500 Da), and then lyophilized to obtain a white solid PCAEB. P .

[0183]

[0184] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0185]

[0186] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0187]

[0188] Example 15

[0189] By replacing PPOBLG-NCA with PLG-NCA, the n-butylamine-initiated polypeptide PAEK was synthesized according to the method described in Example 9. Boc -2.

[0190]

[0191] In PAEK Boc Based on -2, PCAK was synthesized via click chemistry reaction, following the method of Example 10. Boc -2.

[0192]

[0193] In PCAK Boc Based on -2, PCAK-2 was synthesized by deprotection reaction according to the method of Example 11.

[0194]

[0195] In PCAK Boc Based on PCAEK-2, PCAEK-2 was synthesized by quaternization reaction according to the method of Example 12.

[0196]

[0197] Based on PCAEK-2, PCAEK was synthesized according to the method of Example 13. CA -2.

[0198]

[0199] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0200]

[0201] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0202]

[0203] Example 16 ROS-responsive nanoswitch

[0204] Oxaloyl chloride (1.41 g, 11 mmol) and lactic acid (1 g, 11 mmol) were dissolved in tetrahydrofuran (30 mL) under ice bath conditions, and the reaction was carried out for 1 hour. After the reaction was completed, the solvent was removed under vacuum to obtain compound ACC.

[0205]

[0206] PCAEB (30 mg) was dissolved in anhydrous N,N-dimethylformamide (3 mL). Under ice bath conditions, ACC (10 mg) and triethylamine (200 μL) were added, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, the mixture was dialyzed against deionized water for three days (MWCO = 3500 Da), and then lyophilized to obtain a white solid PCAEB. C .

[0207]

[0208] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0209]

[0210] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0211]

[0212] By replacing PLG-NCA with PLG-NCA, the n-butylamine-induced polypeptide PAEB(tBu)-2 was synthesized according to the method in Example 8.

[0213]

[0214] Based on PAEB(tBu)-2, ​​PCAB(tBu)-2 was synthesized by click chemistry according to the method in Example 9.

[0215]

[0216] Based on PCAB(tBu)-2, ​​PCAB-2 was synthesized by deprotection reaction according to the method in Example 10.

[0217]

[0218] Based on PCAB-2, PCAEB-2 was synthesized by quaternization reaction according to the method in Example 11.

[0219]

[0220] Based on PCAEB-2, PCAEB was synthesized via esterification according to the method described in Example 17. C -2.

[0221]

[0222] By replacing cholesterol azide with tocopherol azide, a polypeptide ion channel with tocopherol as the membrane anchoring unit was synthesized using the same method, as shown in the following formula:

[0223]

[0224] By replacing benzo18-crown-6 ether with benzo15-crown-5 ether, a polypeptide ion channel with benzo15-crown-5 ether as the ion transport unit was synthesized using the same method, as shown below:

[0225]

[0226] Test case

[0227] (1) Determine the ion transport efficiency of PCAE(L) and PCAE(DL).

[0228] Synthesized L-type polypeptide PPOBLG initiated by n-butylamine. Taking PPOBLG with a degree of polymerization of 60 as an example, in a glove box, POBLG-NCA (100 mg, 0.315 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and a solution of n-butylamine in N,N-dimethylformamide (53 µL, 0.1 mol / L, [M]0 / [I]0 = 60) was added, and the reaction was carried out at room temperature for 72 hours. The reaction progress was monitored by Fourier transform infrared spectroscopy until the monomer conversion rate reached 100%. After the reaction was completed, the solution was added dropwise to deionized water (100 mL) with vigorous stirring to obtain a white solid. The solution was washed three times with deionized water to remove residual impurities and dried under vacuum to obtain white solid PPOBLG (DP = 60).

[0229] In a glove box, PPOBLG (DP = 60) (30 mg, 0.11 mmol alkynyl group), N,N-dimethyl-3-azidopropylamine (12.4 mg, 0.097 mmol), and azidocholesterol (10 mg, 0.024 mmol) were dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of pentamethyldiethylenetriamine (34 µL, 0.125 mmol) and cuprous bromide (18 mg, 0.125 mmol). The reaction was carried out at room temperature for 48 hours. After the reaction was complete, the mixture was exposed to air to oxidize the excess cuprous bromide, and hydrochloric acid (4 mL, 1 mol / L) was added until the solution changed from blue to yellow. The crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), lyophilized, and yielded a white flocculent solid PCA(L) (DP = 60).

[0230] PCA(L) (DP = 60) (37 mg, 0.09 mmol of tertiary amine group) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and sodium iodide (124 mg, 0.83 mmol) and CE-Br (143 mg, 0.28 mmol) were dissolved in acetonitrile (2 mL). The two mixtures were then reacted at 80 °C for 48 hours. After the reaction was complete, saturated sodium chloride solution (4 mL) was added to the mixture, and the mixture was stirred at room temperature for 4 hours to carry out ion exchange. After the reaction was complete, the crude product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), and then lyophilized to obtain a white solid PCAE(L) (DP = 60).

[0231] The remaining L-type polypeptide ion channels PCAE(L) and DL-type polypeptide ion channels PCAE(DL) were synthesized using the same method.

[0232] LUVs (large single-compartment liposomes, 20 µL) loaded with trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid were added to 4-hydroxyethylpiperazine ethanesulfonic acid buffer (2 mL, pH 7.6, 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 100 mM MCl, M = Li, Na, K, Rb or Cs) and placed in a fluorescence cuvette. The change in fluorescence intensity of the solution over time (λ) was measured using a fluorescence spectrometer. ex =460 nm, λ em =510 nm). At t = 20 s, PCAE(L) or PCAE(DL) with different degrees of polymerization (32 µL, 1 mg / mL) was rapidly added to the system, shaken well, and the fluorescence intensity was monitored. At t = 400 s, Triton X-100 (10 µL, 20%) was added to cleave LUVs. The fluorescence intensity was normalized using the following formula:

[0233] I = (I t - I0) / (I ∞ - I0)

[0234] Among them, I t The fluorescence intensity at time t, I0 and I ∞ These are the initial fluorescence intensity and the final fluorescence intensity after LUV fragmentation, respectively.

[0235] The ion transport efficiencies of PCAA(L) and PCAA(DL) were determined using the method described above. The preparation method of PCAA is similar to that of PCAE, and its structural formula is as follows:

[0236]

[0237] For PCAEK CAand PCAEK SA Potassium ion transport efficiency at pH 6.8 and 7.4, PCAEK CA and PCAEK SA The sample was first pretreated with HCl (1 mol / L) and then added to the LUVs system loaded with HPTS. The change in fluorescence intensity over time was measured according to the method described above.

[0238] PCAE(L) against K + and Na + half-maximal effect concentration (EC50) 50 By K + and Na + After normalizing the fluorescence intensity, the ion transport efficiency was calculated using the Hill equation:

[0239] I = 1 / (1 + (EC 50 / [C] )^m )

[0240] Where [C] represents the concentration of the polypeptide ion channel, and m is a constant obtained from the fitting.

[0241] The ion selectivity of PCAE(L) will be fitted by the following formula:

[0242] S K+ / NA+ =EC 50 [K + ] / EC 50 [Na + ]

[0243] like Figure 12 As shown, PCAE(L) can efficiently K-polymerize when the degree of polymerization is above 40. + Transshipment, and has K + Selectivity is key; PCAE(DL), regardless of its degree of polymerization, cannot perform potassium ion transport. Furthermore, as... Figure 13 As shown, PCAEK CA After acid treatment, efficient K can also be carried out. + transport.

[0244] (2) Validation of membrane anchoring of PCAE (DP = 60)

[0245] 1,2-Dioleoyl-SN-glycerol-3-phosphorylethanolamine (DOPE) (5 mg) was dissolved in dichloromethane (2 mL), and finally Cy5-NHS (5 µL, 5 mg / mL) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by rotary evaporation. Cy5 DOPE was dispersed in ultrapure water at a concentration of 2.5 mg / mL.

[0246] Will Cy5 DOPE (2.5 µL) was added to a LUVs solution (2 mL, 1 mg / mL), and the solution was incubated at room temperature in the dark for 1 hour to prepare Cy5-labeled LUVs. Cy5 LUVs). Then respectively to Cy5 Add to LUVs Cy3 PCAE(L) and Cy3 PCAE(DL) (32 µL, 1 mg / mL, DP = 60). The fluorescence intensity (λ) of the solution was measured using a fluorescence spectrometer. ex = 550 nm, λ em = 560-800 nm). For example... Figure 14 As shown, both PCAE(L, DP = 60) and PCAE(DL, DP = 60) have membrane anchoring capabilities.

[0247] (3) Verification of cell membrane anchoring of PCAE

[0248] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5 (cells / well), incubate overnight at 37°C. Replace the medium with DMEM medium without FBS (fetal bovine serum), and... Cy3 PCAE(L) and Cy3 PCAE (DL) was added to the wells at a final concentration of 10 µg / mL and incubated at 37 °C for 2 hours. After incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and the nuclei were stained with DAPI (4',6-diamidinyl-2-phenylindole, 10 µg / mL). Cells were washed three times with PBS, mounted, and observed and photographed using a CLSM laser confocal microscope. Figure 15 As shown, both PCAE(L) and PCAE(DL) have membrane anchoring capabilities.

[0249] (4) Intracellular potassium ion efflux

[0250] Melanoma B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5Cells were cultured overnight at 37 °C. The medium was replaced with FBS-free DMEM, and PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) were added to the wells at a final concentration of 10 µg / mL. Cells were incubated at 37 °C for 6 hours. After incubation, cells were rinsed three times with PBS. Cells were then incubated for 45 minutes with potassium ion probe EPG-4 AM (300 µL, 3.3 µg / mL) and pluronic® F127 (poloxam, 0.5 μL, 6 wt% dissolved in anhydrous dimethyl sulfoxide). After incubation, cells were rinsed three times with PBS and incubated at 37 °C for another 30 minutes. Subsequently, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI (10 µg / mL), rinsed three times with PBS, mounted, and observed and photographed using CLSM. Figure 16 As shown, PCAE(L, DP = 40, 60, 80) can mediate intracellular potassium ion efflux, while PCAE(DL, DP = 40, 60, 80) cannot.

[0251] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5 (1 cell / well), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.5), and add PCAEK. CA and PCAEK SA Cells were added to wells at a final concentration of 10 µg / mL and incubated at 37 °C for 6 hours. After incubation, cells were washed three times with PBS. Cells were then incubated for 45 minutes with potassium ion probe EPG-4 AM (300 µL, 3.3 µg / mL) and pluronic® F127 (0.5 μL, 6 wt% dissolved in anhydrous dimethyl sulfoxide). After incubation, cells were washed three times with PBS and incubated at 37 °C for another 30 minutes. Subsequently, cells were fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI (10 µg / mL) to the nuclei, washed three times with PBS, mounted, and observed and photographed using CLSM. Figure 17 As shown, PCAEK CA At pH 6.8, it can induce potassium ion efflux from cells, while PCAEK SA It is not possible under either acidic or alkaline conditions.

[0252] (5) Assessment of cellular reactive oxygen species levels

[0253] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5Cells were cultured overnight at 37 °C (cells / well). The medium was replaced with FBS-free DMEM, and PCAE (L, DP = 40, 60, 80) and PCAE (DL, DP = 40, 60, 80) were added to the wells at a final concentration of 10 µg / mL. Cells were incubated at 37 °C for 6 hours. After incubation, cells were rinsed three times with PBS. Cells were then incubated with the reactive oxygen species probe DCFH-DA (2',7'-dichlorodihydrofluorescein, 5 µg / mL, 1 mL) for 25 minutes. After incubation, cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI (10 µg / mL) to the nuclei, rinsed three times with PBS, mounted, and observed and photographed using CLSM. Figure 18 As shown, PCAE(L, DP = 40, 60, 80) can induce cells to produce a large amount of reactive oxygen species, while PCAE(DL, DP = 40, 60, 80) cannot.

[0254] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5 (1 cell / well), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.8), and add PCAEK. CA and PCAEK SA Cells were added to wells at a final concentration of 10 µg / mL and incubated at 37 °C for 6 hours. After incubation, cells were washed three times with PBS. Cells were then incubated with the reactive oxygen species probe DCFH-DA (5 µg / mL, 1 mL) for 25 minutes. After incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI (10 µg / mL) to the nuclei, washed three times with PBS, mounted, and observed and photographed using CLSM. Figure 19 As shown, PCAEK CA At pH 6.8, it can induce the production of reactive oxygen species in cells, while PCAEK SA It is not possible under either acidic or alkaline conditions.

[0255] (6) Detection of mitochondrial membrane potential polarization

[0256] B16F10 cells were seeded into confocal dishes (5 × 10⁻⁶). 5Cells were cultured overnight at 37 °C (cells / plate). The medium was replaced with FBS-free DMEM, and PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) were added to the wells at a final concentration of 10 µg / mL. The cells were incubated at 37 °C for 6 hours. After incubation, the cells were washed three times with PBS. Subsequently, the cells were stained with the JC-1 probe for mitochondrial membrane potential detection for 20 minutes. After staining, the cells were washed three times with PBS, and the cells were observed and photographed using CLSM. Figure 20 As shown, PCAE(L, DP = 40, 60, 80) can induce polarization of the mitochondrial membrane potential, while PCAE(DL, DP = 40, 60, 80) cannot.

[0257] B16F10 cells were seeded into confocal dishes (5 × 10⁻⁶). 5 Cells / plate), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.8), and add PCAEK. CA and PCAEK SA Cells were added to wells at a final concentration of 10 µg / mL and incubated at 37 °C for 6 hours. After incubation, cells were washed three times with PBS. Subsequently, cells were stained with the JC-1 probe for 20 minutes. After staining, cells were washed three times with PBS, observed and photographed using a CLSM scanner. Figure 21 As shown, PCAEK CA At pH 6.8, it can induce polarization of the mitochondrial membrane potential, while PCAEK SA It is not possible under either acidic or alkaline conditions.

[0258] (7) Cytochrome C release

[0259] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5Cells were cultured overnight at 37 °C (cells / well). The medium was replaced with FBS-free DMEM, and PCAE (L, DP = 40, 60, 80) and PCAE (DL, DP = 40, 60, 80) were added to the wells at a final concentration of 10 µg / mL. The cells were incubated at 37 °C for 6 hours. After incubation, the cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with TBS. Subsequently, the cells were permeabilized with Triton X-100 (0.5%, 1 mL) for 10 minutes, blocked with FBS (5% dissolved in PBS) for 30 minutes, and incubated overnight at 4 °C with cytochrome C antibody (1:500 dissolved in Tris-buffered saline TBS). On the second day, cells were rinsed twice with TBST (a buffer formed by adding Tween to TBS), then three times with TBS, and incubated for 1 hour with Cy3-labeled goat anti-rabbit IgG (H+L) (1:400 dissolved in TBS). Cell nuclei were stained with DAPI (5 µg / mL) for 30 minutes. Cells were observed and photographed using CLSM. Figure 22 As shown, PCAE(L, DP = 40, 60, 80) can induce cells to release cytochrome C, while PCAE(DL, DP = 40, 60, 80) cannot.

[0260] B16F10 cells were seeded into 12-well plates containing cell spreaders (5 × 10⁻⁶ cells per well). 5 (1 cell / well), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.8), and add PCAEK. CA and PCAEK SA Cells were added to wells at a final concentration of 10 µg / mL and incubated at 37 °C for 6 hours. After incubation, cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with TBS. Subsequently, cells were permeabilized with Triton X-100 (0.5%, 1 mL) for 10 minutes, blocked with FBS (5% dissolved in PBS) for 30 minutes, and incubated overnight at 4 °C with cytochrome C antibody (1:500 dissolved in TBS). The next day, cells were washed twice with TBST, three times with TBS, and incubated for 1 hour with Cy3-labeled goat anti-rabbit IgG (H+L) (1:400 dissolved in TBS). Cell nuclei were stained with DAPI (5 µg / mL) for 30 minutes. Cells were observed and photographed using CLSM. Figure 23 As shown, PCAEK CA At pH 6.8, it can induce the release of cytochrome C, while PCAEK... SA It is not possible under either acidic or alkaline conditions.

[0261] (8) Detection of apoptosis level

[0262] B16F10 cells were seeded into 12-well plates (5 × 10⁻⁶ cells per well). 5 Cells were cultured overnight at 37 °C (cells / well). The medium was then replaced with FBS-free DMEM, and PCAE(L, DP = 40, 60, 80) and PCAE(DL, DP = 40, 60, 80) were added to the wells at a final concentration of 10 µg / mL. The cells were incubated at 37 °C for 6 hours. The medium was then replaced with DMEM containing 10% FBS, and incubation continued for 18 hours. After incubation, cells were collected, stained with the Annexin V-FITC / PI apoptosis detection kit, and analyzed by flow cytometry. Figure 24 As shown, PCAE(L, DP = 40, 60, 80) can significantly induce apoptosis, while PCAE(DL, DP = 40, 60, 80) cannot.

[0263] B16F10 cells were seeded into 12-well plates (5 × 10⁻⁶ cells per well). 5 (1 cell / well), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.8), and add PCAEK. CA and PCAEK SA Add cells to wells at a final concentration of 10 µg / mL and incubate at 37 °C for 6 hours. Replace the medium with DMEM containing 10% FBS and continue incubation for 18 hours. After incubation, collect cells, stain with Annexin V-FITC / PI apoptosis detection kit, and analyze by flow cytometry. Figure 25 As shown, PCAEK CA It can induce apoptosis at pH 6.8, while PCAEK SA It is not possible under either acidic or alkaline conditions.

[0264] (9) In vitro antitumor efficacy

[0265] B16F10 cells were seeded into 96-well plates (1 × 10⁻⁶). 4 Cells / well were cultured overnight at 37 °C. The medium was then replaced with FBS-free DMEM, and PCAE(L) and PCAE(DL) at final concentrations of 1, 2, 4, 8, 16, 32, and 64 µg / mL were added to the wells, respectively. The cells were incubated at 37 °C for 6 hours. The medium was then replaced with DMEM containing 10% FBS, and incubation continued for 18 hours. Cell viability was determined using the MTT assay, with the viability of untreated cells set at 100%, and the relative viability of cells treated with the polypeptide ion channel was calculated. Figure 26As shown, PCAE (L, DP = 40, 60, 80) can significantly kill B16F10 cells at a concentration of 16 µg / mL.

[0266] B16F10 cells were seeded into 96-well plates (1 × 10⁻⁶). 4 (1 cell / well), incubate overnight at 37°C. Replace the medium with FBS-free DMEM (pH 7.4 or 6.5), and add PCAEK. CA and PCAEK SA Add the solution to the wells at a final concentration of 16 µg / mL and incubate at 37 °C for 6 hours. Replace the medium with DMEM containing 10% FBS and continue incubation for 18 hours. Determine cell viability using the MTT assay, and calculate the relative viability of cells treated with the polypeptide ion channel with the viability of untreated cells as 100%. Figure 27 As shown, PCAEK CA At pH 6.8, it can significantly kill B16F10 cells, while PCAEK SA It is not possible under either acidic or alkaline conditions.

[0267] (10) In vivo antitumor efficacy and biocompatibility studies

[0268] Using B16F10 tumor-bearing female C57BL / 6 mice as a model. When the tumor volume reaches 100 mm... 3 Mice were randomly assigned to four groups of eight mice each, named the PBS group, PCAAK group, etc. CA Group, PCAEK SA Groups and PCAEK CA Group. PBS and PCAAK were administered via tail vein on days 1, 2, 3, and 4, respectively. CA PCAEK SA and PCAEK CA (10 μg polypeptide / kg). Tumor volume was measured daily using calipers, and the mice were weighed. The formula for calculating tumor volume was: Volume = Length × Width. 2 / 2. When the tumor volume reaches 1000 mm 3At the above point, the mice were considered dead, and treatment was terminated, with continued observation of mouse survival. On day 15 of treatment, one mouse from each group was sacrificed, and the heart, liver, spleen, lungs, kidneys, and tumors were collected. These tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), observed under an optical microscope, and subjected to histological analysis. Additionally, tumor tissues were embedded in OCT embedding medium, incubated overnight at -20 °C, and then frozen sectioned. The frozen sections of tumor tissue were stained using a one-step TUNEL apoptosis detection kit, and cell nuclei were stained with DAPI (5 µg / mL). Apoptosis of tumor cells was observed using CLSM. Figures 28-30 As shown, 15 days after administration, PCAEK CA The tumors in the PBS group were significantly suppressed, while those in the PCAAK group were significantly suppressed. CA and PCAEK SA The tumors in this group were not effectively suppressed, and the tumor volume exceeded 1000 mm. 3 This indicates that PCAEK CA It can effectively inhibit tumor growth. Furthermore, PCAEK... CA The survival time of mice in the treatment group was also significantly prolonged. Furthermore, the body weight of mice in all treatment groups did not change significantly during the 15-day observation period. Immunohistochemical sections and immunofluorescence results of tumor tissue showed PCAEK... CA The group exhibited the highest rates of cell necrosis and apoptosis, indicating its excellent in vivo antitumor efficacy. Fifteen days after administration, the heart, liver, spleen, lung, and kidney of mice were collected, and these organs were analyzed by HE staining. The results showed that no significant changes occurred in the major organs after administration (where H represents heart, LI represents liver, SP represents spleen, LU represents lung, and K represents kidney), indicating that the polypeptide ion channel has good biocompatibility.

[0269] (9) Response type of polypeptide ion channel (A represents phosphatase response; R represents ROS response; P represents pH response)

[0270]

[0271] In summary, using n-butylamine as an initiator, a series of pH-responsive polypeptide ion channels were prepared by randomly copolymerizing N-carboxylic anhydride monomers (NCA) containing amino acids or their derivatives through click chemistry, quaternization, deprotection, and amidation reactions. The aim was to simulate the transport process of natural ion channel proteins through the secondary structure transformation of polypeptides, and to further investigate the structure-activity relationship of different secondary structures of polypeptides and the application of polypeptide ion channels in antitumor therapy. The study showed that under normal physiological conditions, due to the electrostatic attraction between the side chains, the polypeptide ion channel PCAEK… CAAs a flexible, random coil structure, the side chain functional groups are arranged in a disordered manner. At this time, the polypeptide ion channel is in a "closed" state and does not have ion transport capabilities. Once the polypeptide ion channel enters the tumor microenvironment, the acidic pH causes the acid-responsive groups to detach. Since the side chains lack electrostatic interactions at this time, the polypeptide ion channel transforms into a rigid α-helix conformation, and the side chain ion transport units are arranged in an orderly manner, which has the ability to induce intracellular potassium ion efflux, thereby achieving the killing of tumor cells.

[0272] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An ion channel, characterized in that, The ion channel comprises a polypeptide backbone and polypeptide side chains, with a structure as shown in formula (I) or (II): ,in: The amino acids forming the polypeptide backbone are L-type amino acids, and R3 is... R4 is the membrane anchoring structure; X is a hydrocarbon group with 0-20 carbon atoms; Y is the linking chain; R1 is a substituted or unsubstituted quaternary ammonium group; and R2 is a benzocrown ether. n is an integer between 40 and 1000, and x is between 0.01 and 0.

99.

2. The ion channel according to claim 1, characterized in that, R4 is selected from , , or .

3. The ion channel according to claim 1 or 2, characterized in that, R1 is R5, R6 and R7 are independently selected from hydrogen or alkyl groups; And / or, the crown ether in R2 is selected from 18-crown-6 ether, 15-crown-5 ether, 21-crown-7 ether or 24-crown-8 ether.

4. The ion channel according to any one of claims 1-3, characterized in that, R3 is , where a is an integer from 0 to 20, b is an integer from 0 to 20, and R5 and R6 are independently selected from hydrogen or alkyl groups.

5. A responsive ion channel, characterized in that, The responsive ion channel comprises a polypeptide backbone and polypeptide side chains, with a structure as shown in one of formulas (IV)-(VII): ,in: The amino acids forming the polypeptide backbone are L-type amino acids, and R3 is... R4 is a membrane anchoring structure, R8 is an anionic group; X is a hydrocarbon group with 0-20 carbon atoms, Y is a linking chain, R1 is a substituted or unsubstituted quaternary ammonium salt group, and R2 is a benzocrown ether; n+y is an integer between 40 and 1000, x is between 0.01 and 0.99, and n / y ≥ 4.

6. The responsive ion channel according to claim 5, characterized in that, R8 is a structure containing a terminal carboxyl group or a terminal phosphate group; R8 includes one of the following structures: 、 、 。 7. The responsive ion channel according to claim 5 or 6, characterized in that, The responsive ion channel is at least pH responsive, phosphatase responsive, or reactive oxygen species responsive.

8. The use of the ion channel according to any one of claims 1-4 or the responsive ion channel according to any one of claims 5-7 in the preparation of ion transport products or drug delivery products.

9. The use of the ion channel according to any one of claims 1-4 or the responsive ion channel according to any one of claims 5-7 in the preparation of diagnostic or therapeutic products.

10. The application according to claim 9, characterized in that, The application includes any of the following: (1) Preparation of tumor diagnostic drugs; (2) Preparation of tumor treatment drugs; (3) Preparation of diagnostic drugs for ion channel diseases; (4) Preparation of drugs for treating ion channel diseases; (5) Preparation of drugs for inflammatory diagnosis; (6) Preparation of anti-inflammatory drugs; (7) Prepare diagnostic drugs for infections, including bacterial infections; (8) Prepare an infection treatment drug, wherein the infection includes bacterial infection.

Images