A pH-responsive water-soluble n-sulfonyl amidine polyamphiphile library, a preparation method and application of n-sulfonyl amidine in conformation regulation

Abstract

The application belongs to the field of polymer synthesis and stimuli-responsive materials, and discloses a pH-responsive water-soluble N-sulfonamidine poly-peptide library, a preparation method and application of N-sulfonamidine in conformation regulation. The water-soluble poly-peptide library mainly has two types: when a homopoly-peptide chain is used as a main chain, N-sulfonamidine containing a glycol fragment or an ionic type is used as a side chain; when a polyethylene glycol block poly-peptide is used as the main chain, the type of the side chain N-sulfonamidine is not limited. The water-soluble poly-peptide has sensitive pH responsiveness, and through pH adjustment, the poly-peptide can be reversibly switched between a 'random coil' and an 'alpha-helix' conformation; through optimization of the substituents on the structure of the N-sulfonamidine, the pKa and protonation ability of the corresponding poly-peptide can be adjusted, so that the pH threshold of the poly-peptide conformation transition is changed.

Description

Technical Field

[0001] This invention belongs to the field of polymer synthesis and stimulus-responsive materials, and relates to a pH-responsive water-soluble N-sulfonylamidine polypeptide library, its preparation method, and the application of N-sulfonylamidine in conformational regulation. Background Technology

[0002] pH-responsive polymers have been widely used in the design of triggered responsive nanomaterials because pH values ​​vary greatly among different tissues and cells. For example, the extracellular environment of tumor cells (pH≈6.5) is more acidic than that of blood (pH≈7.4), while the intracellular / lysosomal environment (pH≈4.5–5.0) is even more acidic. Although many pH-triggered nanomaterials have been successfully developed, most cases focus on responsiveness to intracellular pH, with less attention paid to the development of nanomaterials that can sensitively respond to the acidity inside tumors. Synthetic peptides are a class of protein mimics with rich structural diversity, good biocompatibility, and degradability. Compared with traditional non-structured polymers, they can form highly ordered secondary structures (α-helices and β-sheets) through peptide hydrogen bonds (C=O···HN). The formation of α-helices is mainly caused by intramolecular hydrogen bond interactions. Therefore, side-chain factors that can affect the main chain hydrogen bonding, such as charge, polarity, and hydrogen bonds, will inevitably interfere with the α-helical conformation of peptides, causing conformational changes. This random coil-helix conformational shift of polypeptides is also widely used in various biomedical fields, such as selective cell internalization, gene delivery, antimicrobial materials, and self-assembly.

[0003] Our research group previously reported a novel multicomponent polymerization method for synthesizing N-sulfonylamidine (SAi) polymers, which unexpectedly exhibit zwitterionic properties in response to pH changes (Sci. China Chem. 2022, 5, 1798-806). It is generally believed that the amidine group, as a "base," can be protonated, carrying a positive charge under neutral or acidic conditions and a neutral charge under alkaline conditions. However, due to the electron-withdrawing properties of the sulfonyl group, SAi exhibits properties similar to a "weak acid," rather than a "base." This characteristic inspired us to further explore the relationship between its structure and pKa value, expanding its potential in designing pH-responsive polymers.

[0004] Therefore, we adopted a one-step multi-component reaction as a post-polymerization modification strategy to introduce N-sulfonylamidine onto the side chains of polypeptides, obtaining water-soluble N-sulfonylamidine-based polypeptides. By optimizing the structure of N-sulfonylamidine, we can regulate the pKa and protonation ability of the polypeptides, thereby achieving a reversible transition from random coil to α-helix configuration of the polypeptides within the pH range of 6.2-7.0. This pH adjustment range matches the acidity of the tumor microenvironment, and this type of polypeptide has great potential in stimulus-responsive biomedical materials. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing a water-soluble polypeptide library modified with N-sulfonylamidine side chains.

[0006] The technical solution of this invention:

[0007] An N-sulfonylamidine water-soluble polypeptide has the following structure:

[0008]

[0009] in:

[0010] R is: R in the left equation 1 and R 2 It is an oligoethylene glycol, polyethylene glycol, or a water-soluble alkyl substituent with a quaternary ammonium ion terminus; R in the formula on the right 1 and R 2 It is phenyl or C1-C 10 The alkyl substituents; n represents the degree of polymerization, 20≤n≤500.

[0011] A method for preparing an N-sulfonylamidine water-soluble polypeptide, comprising the following steps:

[0012]

[0013] In a glove box, alkynyl polypeptides (homopolymer alkynyl polypeptides or polyethylene glycol block alkynyl polypeptides, calculated as alkynyl groups), amine compounds, cuprous iodide, and triethylamine are dissolved in dry N,N-dimethylformamide (DMF) at a molar ratio of 1:(2-4):(0.1-1):(0.5-5), wherein the concentration of alkynyl polypeptides is 0.1-0.5M. Then, 1-3 molar equivalents of p-toluenesulfonyl azide relative to the alkynyl group are slowly added dropwise to the above solution. After stirring at room temperature for 12-48 hours, the solution is precipitated in ten times its volume of diethyl ether and washed three times with diethyl ether. After vacuum drying, the crude product is reconstituted with DMF, and then EDTA-Na2 solution is added. The solution is dialyzed with deionized water for 24-36 hours and freeze-dried to obtain N-sulfonylamidine water-soluble polypeptides.

[0014] A second objective of this invention is to provide a method for regulating the conformational changes of polypeptides using pH-responsive N-sulfonylamidinium.

[0015] The beneficial effects of this invention are:

[0016] (1) The present invention constructs a novel N-sulfonylamidine side chain modified polypeptide library through a simple one-step multi-component synthesis method. Compared with traditional N-sulfonylamidine structured polymers, the polypeptide has excellent water solubility and tunable secondary structure.

[0017] (2) This invention proposes a new method for regulating the pKa and protonation ability of water-soluble polymers by the side chain N-sulfonylamidine structure.

[0018] (3) This invention proposes a new method for regulating the secondary structure of polypeptides using a sulfonylamidine structure, which can reversibly regulate conformational changes by adjusting pH.

[0019] (4) Compared with traditional pH-responsive polypeptides, this type of polypeptide has a finer pH adjustment range and can undergo conformational changes at pH levels close to the tumor microenvironment, which has great application potential in the biomedical field. Attached Figure Description

[0020] Figure 1 This is the pH titration curve of PPLG-EG2 in Example 4;

[0021] Figure 2 These are the pH titration curves of PPLG-Bu and PPLG-Hex in Example 4;

[0022] Figure 3 It is PEG-PPLG in Example 4 20 pH titration curve of PA;

[0023] Figure 4 It is PEG-PPLG in Example 4 20 -pH titration curve of HA;

[0024] Figure 5 It is PEG-PPLG in Example 4 20 pH titration curve of -DA;

[0025] Figure 6 It is PEG-PPLG in Example 4 40 pH titration curve of PA;

[0026] Figure 7 It is PEG-PPLG in Example 4 40 -pH titration curve of HA;

[0027] Figure 8 It is PEG-PPLG in Example 4 40 -DA titration curve;

[0028] Figure 9 This is the helicity variation curve of PPLG-EG2 at different pH values ​​in Example 5;

[0029] Figure 10 These are the helicity variation curves of PPLG-Bu and PPLG-Hex at different pH values ​​in Example 5;

[0030] Figure 11 It is PEG-PPLG in Example 5 20 -PA, PEG-PPLG 20 -HA and PEG-PPLG 20 -Helicity variation curves of DA at different pH levels;

[0031] Figure 12 It is PEG-PPLG in Example 5 40 -PA, PEG-PPLG 40 -HA and PEG-PPLG 40 -DA helicity variation curve at different pH levels. Detailed Implementation

[0032] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0033] Example 1: Synthesis of alkynyl polypeptide (PPLG-EG2)

[0034] γ-Prolyl-L-glutamic acid-N-carboxylic acid intracyclic anhydride (PLG-NCA) was synthesized according to previous reports (Macromol. Rapid Commun. 2010, 31, 991-997).

[0035] In a glove box, PLG-NCA (170 mg, 0.805 mmol) was dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF), followed by the addition of 32 μL (0.5 M) of DMF stock solution of n-hexylamine. After stirring at room temperature for 2 days, the solution was precipitated into 10 volumes of diethyl ether, washed three times with ether, and dried under vacuum overnight to give a white precipitate of polyamino acid PPLG (70% yield). Its molecular weight was determined by GPC: M w =12221; M n =10465; according to 1 H NMR analysis yielded a polymerization value of 54.

[0036] Example 2: Synthesis of N-sulfonylamidinyl polypeptide

[0037]

[0038] The synthesis method of PPLG-EG2 is as follows: In a glove box, PPLG (15 mg, 87.7 μmol), 2-(2-methoxyethoxy)ethylamine (32 mg, 263 μmol), cuprous iodide (3.3 mg, 17.5 μmol), and triethylamine (44 mg, 438 μmol) were dissolved in 500 μL of dry DMF, and p-toluenesulfonyl azide (47 mg, 240 μmol) was added dropwise to the solution. The solution was stirred at room temperature for 12 h, and then precipitated three times with 10 times its volume of diethyl ether. After vacuum drying, the crude product was reconstituted with DMF, and EDTA-Na2 solution was added. Dialysis with deionized water was performed for 24 h, with the deionized water changed every 6 h. After freeze-drying, a white flocculent precipitate PPLG-EG2 was obtained (yield 50%). 1 1H NMR analysis showed that the modification rate of SAi was 60%.

[0039] Synthesis of PPLG-Bu and PPLG-Hex: The synthesis method is as described in Example 1, wherein the amine is replaced with the corresponding amine compound: PEG. 350 -NH2, 4-amino-N,N,N-trimethyl-1-butanylamine, 4-amino-N,N,N-trimethyl-1-hexylamine. The yields of PPLG-Bu and PPLG-Hex were 56% and 58%, respectively, corresponding to sulfonamide modification rates of 47% and 59%.

[0040] Example 3: Synthesis of N-sulfonylamidinidine polypeptides PEG-PPLG-PA, PEG-PPLG-HA and PEG-PPLG-DA

[0041] In the glove box, mPEG 5k -NH2 (400 mg, 75.8 μmol) was dissolved in 3 mL of dry DMF, and PLG-NCA in 1 mL of DMF (400 mg, 1.895 mmol) was added. After stirring at room temperature for two days, the solution was precipitated into 10 times the volume of ice-cold diethyl ether / n-hexane (v:v = 1:1). The precipitate was washed three times with diethyl ether. After vacuum drying, a white solid was obtained, which was PEG-PPLG. 1 ¹H NMR analysis showed that the degree of polymerization of PEG-PPLG was 20. They were named PEG-PPLG. 20 .

[0042] PEG-PPLG 40 The synthesis method of PEG-PPLG 20 Same, but uses 40 equivalent PLG-NCA.

[0043]

[0044] PEG-PPLG20 -PA is synthesized as follows: PEG-PPLG 20 (100 mg, acetylene group = 0.257 mmol), aniline (72 mg, 0.77 mmol), CuI (9.8 mg, 51.4 μmol), and TEA (130 mg, 1.285 mmol) were dissolved in 2.5 mL of dry DMF. Then, p-toluenesulfonyl azide (102 mg, 515 μmol) was added dropwise to the solution. After stirring for 12 h, the solution was precipitated three times with 10 times its volume of diethyl ether and dried under vacuum. The crude product was dissolved in DMF, and EDTA-Na2 solution was added. The solution was dialyzed against deionized water for 24 h. The deionized water was changed every 6 h. After lyophilization, a white solid PEG-PPLG was obtained. 20 -PA. Under similar conditions, by changing the type of amine, PEG-PPLG was prepared using n-hexylamine as a raw material. 20 -HA, PEG-PPLG prepared from diethylamine 20 -DA.

[0045] For PEG-PPLG 40 -PA, PEG-PPLG 40 -HA and PEG-PPLG 40 The synthesis method for -DA is the same as above, wherein PEG-PPLG is used. 40 The following information pertains to the polypeptide obtained in Example 3, which served as the starting material:

[0046] Table 1. Degree of polymerization and sulfonylamidine modification rate of polypeptides

[0047]

[0048] Example 4: pH titration curve and pKa determination of N-sulfonylamidinium polypeptide

[0049] The sulfonylamidine peptide was dissolved in HCl solution (1 mg / mL, 6 mL), and titrated with small volumes (5 μL increments) of 0.1 M NaOH solution, with the pH value measured in real time using a pH meter. The pH value was correlated with the volume of NaOH solution to obtain the corresponding first derivative. The extreme points of the first derivative were used as the start and end points of protonation to calculate the degree of protonation of the SAi peptide. The pH corresponding to 50% protonation was the pKa value.

[0050] The pH titration curve of PPLG-EG2 is as follows: Figure 1 As shown;

[0051] The pH titration curves of PPLG-Bu and PPLG-Hex are as follows: Figure 2 As shown;

[0052] PEG-PPLG 20-PA pH titration curve as shown Figure 3 As shown;

[0053] PEG-PPLG 20 -HA pH titration curve as follows Figure 4 As shown;

[0054] PEG-PPLG 20 -DA pH titration curve as follows Figure 5 As shown;

[0055] PEG-PPLG 40 -PA pH titration curve as shown Figure 6 As shown;

[0056] PEG-PPLG 40 -HA pH titration curve as follows Figure 7 As shown;

[0057] PEG-PPLG 40 -DA pH titration curve as follows Figure 8 As shown.

[0058] Example 5: Characterization of pH-responsive conformational transition of N-sulfonylamidinyl polypeptide

[0059] N-sulfonylamidine polypeptides were dissolved in an aqueous solution, and the pH was adjusted by adding a specific volume of concentrated HCl or NaOH and measured with a pH meter. The solution was then placed in a quartz cell for circular dichroism (CD) spectroscopy, and its helicity was calculated using the formula: [θ] λ =(MRW×θ) λ ) / (d×c), helicity = (-[θ] 222 +3000) / 39000, where MRW is the average residual mass; θ λ λ is the observed ellipticity at wavelength λ (i.e., 222 nm); d is the path length (mm); and c is the concentration (mg / mL).

[0060] The curves showing the change in helicity of PPLG-EG2 at different pH values ​​are as follows: Figure 9 As shown;

[0061] The helicity variation curves of PPLG-Bu and PPLG-Hex at different pH values ​​are shown below. Figure 10 As shown;

[0062] PEG-PPLG 20 -PA, PEG-PPLG 20 -HA and PEG-PPLG 20 -DA helicity variation curves at different pH levels are as follows: Figure 11 As shown;

[0063] PEG-PPLG 40 -PA, PEG-PPLG 40 -HA and PEG-PPLG 40 -DA helicity variation curves at different pH levels are as follows: Figure 12 As shown.

Claims

1. A kind N - A sulfonylamidine water-soluble polypeptide, characterized in that, Should N The structure of sulfonylamidine water-soluble polypeptides is shown in Formula 1 or Formula 2 below: Formula 1 Formula 2 in: R is: In Equation 1, R 1 and R 2 It is an oligoethylene glycol, polyethylene glycol, or a water-soluble alkyl substituent with a quaternary ammonium ion terminus; R in Formula 2 1 and R 2 It is phenyl or C1-C 10 The alkyl substituent; n represents the degree of polymerization, 20≤n≤500.

2. A device as described in claim 1 N A method for preparing sulfonylamidine water-soluble polypeptides, characterized in that, The steps are as follows: ； In a glove box, alkynyl polypeptides, amine compounds, cuprous iodide, and triethylamine were dissolved in a dry solution at a molar ratio of 1:(2-4):(0.1-1):(0.5-5). N , N In dimethylformamide, the concentration of the alkynyl polypeptide is 0.1-0.5 M; then, 1-3 molar equivalents of p-toluenesulfonyl azide relative to the alkynyl group in the alkynyl polypeptide are slowly added dropwise to the above solution. After stirring at room temperature for 12-48 h, the solution is precipitated in ten times its volume of diethyl ether and washed three times with diethyl ether. After vacuum drying, the crude product is... N , N -Reconstitute with dimethylformamide, add disodium ethylenediaminetetraacetate solution, dialyze with deionized water for 24-36 h, and freeze-dry to obtain N -Sulfoamide water-soluble polypeptide; The alkynyl polypeptide is a homopolymer alkynyl polypeptide or a polyethylene glycol block alkynyl polypeptide.

3. The one described in claim 1 N - Sulfonylamidine water-soluble polypeptides are used to precisely respond to pH-reversibly regulated conformational changes.

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