Thermosensitive amphiphilic dendritic polypeptide and preparation method thereof

Abstract

The application discloses a preparation method of a temperature-sensitive amphiphilic dendritic polypeptide, and the amphiphilic dendritic polypeptide can realize visible light regulation and chiral assembly. The structural formula of the amphiphilic dendritic polypeptide is shown in the description. Wherein, n is 1-4, P is 1-4, and X is OEt or OMe. The dendritic alkoxy ether element includes but is not limited to three arms, four arms or six arms. The amphiphilic dendritic polypeptide exhibits a suitable hydrophilic-hydrophobic balance, realizes effective supramolecular assembly in a water environment, and the dendritic alkoxy ether element endows the assembly with characteristic temperature-sensitive behavior. The regulation process has the characteristics of visible light activation, reversible transformation and biological friendliness, and thus has potential application value in the fields of biological medicine, chiral detection, chiral optics and chiral catalysis.

Description

Technical Field

[0001] This invention belongs to the field of intelligent amphiphilic chiral molecules, and relates to a class of thermosensitive amphiphilic dendritic peptides and their preparation methods. The thermosensitive amphiphilic dendritic peptides of this invention can be regulated by visible light in aqueous solution for chiral assembly. Technical Background

[0002] Supramolecular chirality is ubiquitous in nature. For example, the three-dimensional ordered structure of proteins, the complex micro- and nano-structures of nucleic acids, and the specific recognition channels of cells provide the basic units of life activities and determine the diversity of life. Researchers are committed to endowing molecules with intelligent response characteristics to external stimuli through rational molecular design, so as to explore and simulate interesting phenomena in nature and better realize the transmission and amplification of supramolecular chirality and its effective regulation. Compared with other external stimuli, photostimulation response has a series of advantages such as non-invasiveness and precise tunable response range, and has received widespread attention. Traditional photo-response units such as azobenzene, azopyrazole, spiropyran, and cinnamic acid are usually excited by ultraviolet light and have low fatigue strength, thus limiting their further application in the field of biomaterials (Lerch M., Szymanski W., et al. The (photo)chemistry of Stenhouse photoswitches: guiding principles and system design[J]. Chem. Soc. Rev., 2018, 47(6): 1910-1937.). Donor-Acceptor Stenhouse Adducts (DASA) are a novel type of molecular photoswitch with advantages such as simple synthesis, visible light activation, high sensitivity, and excellent fatigue resistance (Helmy S., Leibfarth F A., et al. Photoswitching using visible light: a new class of organic photochromic molecules[J]. J. Am. Chem. Soc., 2014, 136(23): 8169-8172.). With visible light irradiation, the molecule gradually transforms from a conjugated triene structure to a cyclopentanone ionic structure, resulting in significant changes in molecular color, hydrophilicity / hydrophobicity, and size. However, the cyclopentanone ionic structure is thermodynamically unstable, and therefore, under light-shielded and heated thermal relaxation conditions, a reversible transition to a conjugated triene structure often occurs. However, the reversible isomerization of first-generation DASA in polar solvent water is very challenging. Meanwhile, most reported studies on the photo-irradiated regulation of supramolecular chiral assembly are often conducted in organic solvent environments and excited by ultraviolet light. How to achieve visible light-controlled supramolecular chiral assembly in a highly polar water environment has become an urgent technical problem to be solved. Summary of the Invention

[0003] To address the problems of existing technologies, the present invention aims to overcome the shortcomings of existing technologies and provide a thermo-sensitive amphiphilic dendritic polypeptide and its preparation method, enabling visible light-regulated chiral assembly. In this type of amphiphilic dendritic polypeptide, the DASA motif undergoes reversible hydrophilic-hydrophobic transformation through visible light irradiation isomerization, exhibiting a suitable hydrophilic-hydrophobic balance and achieving effective supramolecular assembly in aqueous solution. The dendritic alkoxy ether motif imparts characteristic thermo-sensitive behavior to the assembled structure. Below the phase transition temperature, the DASA motif transforms from a purple conjugated hydrophobic structure to a colorless, charged hydrophilic isomer upon visible light irradiation, inducing the deconstruction of the assembly. Above the phase transition temperature, the hydrophobic microenvironment formed by the collapse of the dendritic alkoxy ether can drive the DASA motif to effectively revert to the hydrophobic isomer under light-shielded heating in a highly polar aqueous environment, achieving partial reconstruction of the assembly.

[0004] To achieve the above-mentioned objectives, the present invention adopts the following inventive concept:

[0005] This invention constructs a dendritic polypeptide through an amidation reaction of a dendritic alkoxy ether benzylamino group and a proline sequence polypeptide, and then obtains an amphiphilic dendritic polypeptide with visible light-regulated chiral assembly via imine nucleophilic attack on a DASA receptor. The specific reaction formula is as follows:

[0006]

[0007] Wherein: n = 1–4, P = 1–4, X is OEt or OMe, and the dendritic alkoxy ether moiety includes, but is not limited to, three-armed, four-armed, or six-armed. The concept of this invention is not limited to the above-mentioned temperatures and reaction times for amidation and imine nucleophilic attack on DASA receptors; anything that solves the technical problem of this invention falls under the technical principles of this invention.

[0008] Based on the above inventive concept, the present invention adopts the following technical solution:

[0009] A thermosensitive amphiphilic dendritic polypeptide, the molecular structure of which is as follows:

[0010]

[0011] Where: n = 1 to 4, P = 1 to 4, X is OEt or OMe, and the dendritic alkoxy ether unit includes, but is not limited to, three-armed, four-armed or six-armed.

[0012] As a preferred embodiment of the present invention, the thermosensitive amphiphilic dendritic polypeptide is constructed by amidation of a dendritic alkoxy ether moiety and a proline sequence polypeptide. Then, through imine nucleophilic attack on the DASA receptor, the dendritic alkoxy ether and DASA moiety are linked to the chiral polypeptide of the proline sequence. This thermosensitive amphiphilic dendritic polypeptide molecule achieves chiral assembly in an aqueous environment driven by hydrophilicity and hydrophobicity. Furthermore, by isomerizing the DASA moiety under visible light irradiation, the amphiphilicity of the dendritic polypeptide changes, thereby achieving reversible regulation of chiral assembly. The amphiphilic dendritic polypeptide exhibits thermosensitive properties in aqueous solution.

[0013] As a preferred technical solution of the present invention, the amphiphilic dendritic peptides of the present invention can achieve chiral assembly and chiral amplification in an aqueous environment.

[0014] As a preferred technical solution of the present invention, below the phase transition temperature, when the DASA unit is irradiated with visible light, its conjugated hydrophobic structure is transformed into a colorless, charged hydrophilic isomer, inducing the assembly to deconstruct.

[0015] Above the phase transition temperature, the hydrophobic microenvironment formed by the collapse of dendritic alkoxy ethers drives the DASA motif to revert to the hydrophobic isomer in a polar aqueous environment through light-shielded heating, thereby reconstructing the assembly.

[0016] A method for preparing the thermo-sensitive amphiphilic dendritic polypeptide of the present invention includes the following steps:

[0017] a. Amideation process:

[0018] Boc-(Pro)4-OH was prepared by reacting a proline-sequenced tetrapeptide with LiOH·H2O.

[0019] Then, Boc-(Pro)4-OH was dissolved in dry DCM, and 1-hydroxybenzotriazole was added; the dendritic alkoxy ether unit was dissolved in dry DCM, and diisopropylethylamine was added; the mixture was purged and evacuated with high-purity nitrogen at least three times; after reacting in an ice-salt bath for at least 30 min, the reaction mixtures were mixed, purged and evacuated with high-purity nitrogen at least three times, and reacted again in an ice-salt bath for at least 30 min; then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the product mixture, and the mixture was allowed to cool naturally to room temperature overnight; the reaction was stopped after TLC to confirm completion, and the mixture was washed at least three times with saturated sodium bicarbonate solution and potassium bisulfate solution (at least 10 wt.%). The organic phase was dried over anhydrous magnesium sulfate, filtered to evaporate the solvent, and purified by column chromatography to obtain the intermediate product Et-(Pro). n -Boc, its structure is:

[0020]

[0021] Where: n = 1 to 4, P = 1 to 4, X is OEt or OMe, and the dendritic alkoxy ether unit includes, but is not limited to, three-armed, four-armed or six-armed;

[0022] b. The process of imine nucleophilic attack on DASA receptors:

[0023] The Et-(Pro) prepared in step a) n -Boc was dissolved in dry DCM, and an ethyl acetate solution of hydrochloric acid was added under ice-salt bath conditions. The reaction was carried out at a temperature not higher than -15°C for at least 12 hours. The reaction was stopped after TLC to confirm complete reaction. The ethyl acetate was removed by rotary evaporation, and saturated sodium bicarbonate solution was added dropwise until no more bubbles were produced. Then, sodium hydroxide solution with a mass percentage concentration of not less than 10 wt.% was added to adjust the pH of the mixture to 10. The mixture was lyophilized to remove water, and DCM was added to the resulting solid to dissolve it completely. Anhydrous magnesium sulfate was added and dried. After filtration, the solvent was evaporated. The resulting product was dissolved in dry tetrahydrofuran solution, and a DASA acceptor was added. The solution immediately turned deep purple. The reaction was carried out overnight under a dry nitrogen atmosphere at a temperature not lower than 40°C. After the reaction was complete, the solvent was evaporated, and the product was purified by column chromatography to obtain the target product Et-(Pro). n -DASA. Its structural formula is:

[0024]

[0025] Where: n = 1 to 4, P = 1 to 4, X is OEt or OMe, and the dendritic alkoxy ether unit includes, but is not limited to, three-armed, four-armed or six-armed.

[0026] As a preferred embodiment of the present invention, in step a, the molar ratio of Boc-(Pro)4-OH, 1-hydroxybenzotriazole, dendritic alkoxy ether moiety, diisopropylethylamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:2:4:4.

[0027] As a preferred embodiment of the present invention, in step a, the molar ratio of the proline sequence tetrapeptide and LiOH·H2O is 1:1.

[0028] As a preferred embodiment of the present invention, in step a, the dendritic alkoxy ether moiety is dendritic alkoxy ether benzylamine.

[0029] As a preferred embodiment of the present invention, in step b, the molar ratio of the product prepared using Et-(Pro)4-Boc to the DASA receptor is 1:2.

[0030] This invention grafts dendritic alkoxy ethers and DASA motifs onto chiral polypeptides containing proline sequences via amidation and imine nucleophilic attack on DASA donors. This allows for chiral assembly of the molecules in an aqueous environment, driven by hydrophilicity and hydrophobicity. Upon exposure to visible light, the conjugated hydrophobic structure of the DASA motif transforms into a colorless, charged hydrophilic isomer, inducing the deconstruction of the assembly. Above the phase transition temperature, the hydrophobic microenvironment created by the collapse of the dendritic alkoxy ether drives the effective reversion of the DASA motif to the hydrophobic isomer under light-shielded heating in a highly polar aqueous environment, thus achieving assembly reconstruction.

[0031] Compared with the prior art, the present invention has the following obvious and prominent substantive features and significant advantages:

[0032] 1. The present invention introduces a dendritic alkoxy ether motif, which gives the amphiphilic dendritic polypeptide good water solubility and can utilize the dehydration collapse of alkoxy ethers above the phase transition temperature to provide a hydrophobic microenvironment for the molecule.

[0033] 2. The amphiphilic dendritic peptides of the present invention exhibit a suitable hydrophilic-hydrophobic balance, enabling supramolecular assembly in an aqueous environment;

[0034] 3. The amphiphilic dendritic peptides of the present invention utilize visible light irradiation to achieve effective regulation of chiral assembly, and can achieve partial reconstruction of the assemblies in a highly polar water environment through light-shielded heating above the phase transition temperature.

[0035] 4. The amphiphilic dendritic peptides of the present invention have good biocompatibility, visible light-activated reaction characteristics and effective reversibility, giving them potential for application in the field of biomaterials. Attached Figure Description

[0036] Figure 1 The intermediate product Et-(Pro)4-Boc prepared in Example 1 of this invention 1 H NMR spectrum.

[0037] Figure 2 Et-(Pro)4-DASA prepared in Example 1 of this invention 1 H NMR spectrum.

[0038] Figure 3 This is an atomic force microscope (AFM) image of the Et-(Pro)4-DASA chiral assembly in Embodiment 2 of the present invention.

[0039] Figure 4 This is the circular dichroism (CD) spectrum of Et-(Pro)4-DASA as a function of visible light irradiation time in Embodiment 2 of the present invention.

[0040] Figure 5This is the circular dichroism (CD) spectrum of the reversibly recoverable Et-(Pro)4-DASA chiral assembly in Embodiment 2 of the present invention. Detailed Implementation

[0041] For the synthesis of dendritic alkoxy ether units, please refer to: Li, W., Zhang, A., et al. Thermoresponsive dendronized polymers with tunable lower critical solution temperatures[J]. Chem. Commun., 2008, 142(43): 5523-5525.

[0042] The above solution will be further described below with reference to specific embodiments. The preferred embodiments of the present invention are described in detail below:

[0043] Example 1

[0044] In this embodiment, a type of visible light-regulated chiral assembly of amphiphilic dendritic peptides and its preparation method are described, with the following specific steps:

[0045] a. Amide synthesis of Et-(Pro)4-Boc

[0046] Proline tetrapeptide (1 g, 1.92 mmol) was dissolved in a mixed solvent of 50 mL methanol and 10 mL water. LiOH·H2O (0.72 g, 19.2 mmol) was added under ice bath conditions, and the mixture was stirred for 4 h. The reaction was stopped when the reaction was confirmed to be complete by TLC plate. The pH was adjusted to weakly acidic with potassium hydrogen sulfate (10 wt.%), and the mixture was washed three times with saturated brine. The organic phase was dried with anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain Boc-(Pro)4-OH.

[0047] Dissolve Boc-(Pro)4-OH (0.9 g, 1.58 mmol) in 30 mL of dry DCM, add 1-hydroxybenzotriazole (HOBt) (0.22 g, 1.58 mmol), dissolve dendritic alkoxy ether benzylamine (2.1 g, 3.16 mmol) in 30 mL of dry DCM, add diisopropylethylamine (DiPEA) (0.82 g, 6.32 mmol), purge with high-purity nitrogen three times, reacting for 30 min each time under ice-salt bath conditions, then mix the reaction solutions and purge with high-purity nitrogen. After evacuating the air three times and continuing the reaction under ice-salt bath conditions for 30 min, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (1.21 g, 6.32 mmol) was added to the system, and the mixture was allowed to cool naturally to room temperature and reacted overnight. The reaction was stopped after TLC plate detection to confirm its completeness. The mixture was washed three times successively with saturated sodium bicarbonate solution and 10 wt.% potassium bisulfate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated. The intermediate product Et-(Pro)4-Boc was obtained by column chromatography purification.

[0048] Physical property characterization tests were performed on the intermediate products:

[0049] 1 ¹H NMR (d6-DMSO): δ=1.18-1.22(t,9H,H-Boc),1.31-1.38(m,9H,CH3),1.63-2.84(m,16H,CH2),3.20-3.90(m,44H,CH2),4.10-4.67(m,10H,CH+CH2),6.56(s,2H,CH),8.36(s,1H,NH); The product is Et-(Pro)4-Boc.

[0050] b. The process of imine nucleophilic attack on DASA receptors

[0051] Et-(Pro)4-Boc (1.17 g, 1.04 mmol) was dissolved in 25 mL of dry DCM. An ethyl acetate solution of HCl was added under ice-salt bath conditions, and the reaction was carried out at -15 °C for 12 h. The reaction was stopped after TLC to confirm completion. The ethyl acetate was removed by rotary evaporation, and saturated sodium bicarbonate solution was added dropwise until no more bubbles were produced. A small amount of 10% sodium hydroxide solution was added to adjust the pH to 10. The mixture was lyophilized to remove water. DCM was added to the resulting solid and dissolved completely. Anhydrous magnesium sulfate was added and dried. The solvent was evaporated after filtration. The resulting product (0.5 g, 0.57 mmol) was dissolved in dry tetrahydrofuran solution. A DASA acceptor (0.43 g, 1.14 mmol) was added, and the solution immediately turned deep purple. The reaction was carried out overnight at 40 °C under a dry nitrogen atmosphere. After the reaction was complete, the solvent was evaporated, and the product was purified by column chromatography to obtain the target product Et-(Pro)4-DASA.

[0052] Physical property characterization tests were performed on the target product:

[0053] 1 H NMR(d6-DMSO): δ=0.75-0.9(t,6H,CH3),1.15-1.40(m,20H,CH2),1.31-1.38(m,9H,CH3),1 .63-2.84(m,16H,CH2),3.52-3.83(m,43H,CH2),3.93-4.05(m,4H,CH2),3.96-4.12(m,3H,C The product is Et-(Pro)4-DASA, with the following parameters: H+CH2), 4.17-4.24 (m,6H,CH2), 4.32-4.68 (m,2H,CH), 4.87 (m,1H,OH), 5.12-6.23 (m,2H,CH), 6.61-6.85 (m,2H,CH), 6.83-7.85 (m,2H,CH), and 7.8-8.4 (m,1H,NH).

[0054] Example 2:

[0055] In this embodiment, the visible light-controlled chiral assembly and reversible reversibility of Et-(Pro)4-DASA in aqueous solution were tested. The specific steps are as follows:

[0056] a. Atomic force microscopy (AFM) morphology analysis of Et-(Pro)4-DASA chiral assemblies

[0057] The prepared Et-(Pro)4-DASA aqueous solution was added dropwise to the mica substrate, with a solution concentration of 0.02 mg·mL⁻¹. -1After the solvent evaporated, AFM testing was performed. In the aqueous solution, the molecules exhibited obvious aggregation and assembly behavior, forming microspheres with an average diameter of 426±10 nm and an average height of 35±1 nm.

[0058] b. Circular dichroism (CD) spectroscopy of visible light-modulated Et-(Pro)4-DASA chiral assemblies

[0059] Et-(Pro)4-DASA was prepared at a concentration of 1.5 mg / mL. -1 The effect of illumination time on chiral signals was tracked using circular dichroism spectroscopy at room temperature in aqueous solution. Cotton peaks appeared at 205 nm and 258 nm, originating from the absorption of the peptide's amide bond. A relatively obvious Cotton peak also appeared at 577 nm. Since the peptide showed no absorption around 577 nm, it can be determined that the absorption originated from the hydrophobic triene structure, proving that chiral transfer was successfully achieved. With prolonged illumination time, the Cotton peak at 577 nm gradually weakened and eventually disappeared, and this weakening and disappearance was synchronous with the weakening and disappearance of the corresponding ultraviolet absorption peak. This demonstrates that under illumination, the DASA motif transforms from a purple conjugated hydrophobic structure to a colorless, charged hydrophilic structure, causing the assembly driving force of the molecule to disappear, thus leading to the disassembly of the assembly and the disappearance of the chiral signal.

[0060] c. Reversible circular dichroism (CD) spectroscopy of Et-(Pro)4-DASA chiral assembly

[0061] When a colorless Et-(Pro)4-DASA solution is heated above its phase transition temperature under light exposure, the hydrophobic microenvironment formed by the collapse of the dendritic alkoxy ether drives the effective recovery of the DASA moiety to the hydrophobic isomer in a highly polar aqueous environment under light-protected heating. After the UV recovery rate reaches 65%, the chiral signal recovers by 50%.

[0062] In summary, a class of amphiphilic dendritic peptides capable of visible-light-regulated chiral assembly was successfully constructed by grafting dendritic alkoxy ethers and DASA motifs onto chiral peptides with proline sequences via amidation and imine nucleophilic attack on the DASA receptor. The DASA motif in these amphiphilic dendritic peptides can undergo reversible hydrophilic-hydrophobic transitions through visible-light irradiation isomerization, exhibiting a suitable hydrophilic-hydrophobic balance and enabling efficient supramolecular assembly in aqueous solutions. The dendritic alkoxy ether motif endows the assemblies with characteristic thermosensitive behavior. Below the phase transition temperature, the DASA motif transforms from a purple, conjugated hydrophobic structure to a colorless, charged hydrophilic isomer upon visible-light irradiation, inducing the disassembly of the assembly. Above the phase transition temperature, the hydrophobic microenvironment formed by the collapse of the dendritic alkoxy ether drives the DASA motif to effectively revert to its hydrophobic isomer under light-shielded heating in a highly polar aqueous environment, achieving partial reconstruction of the assembly. Its regulation process is characterized by visible light activation, reversible transformation, and bio-friendly properties, thus having potential application value in fields such as biomedicine, chiral detection, chiral optics, and chiral catalysis.

[0063] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made according to the purpose of the invention. Any changes, modifications, substitutions, combinations or simplifications made based on the spirit and principle of the technical solution of the present invention shall be equivalent substitutions. As long as they meet the purpose of the invention and do not deviate from the technical principle and inventive concept of the thermosensitive amphiphilic dendritic macromolecule based on tetraphenylethylene and its preparation method, they shall fall within the protection scope of the present invention.

Claims

1. A thermo-sensitive amphiphilic dendritic polypeptide, characterized in that: Its molecular structural formula is: Where: n = 4, p = 3, X is OEt.

2. A method for preparing the thermo-sensitive amphiphilic dendritic polypeptide according to claim 1, characterized in that: It includes the following steps: a. Amideation process: Boc-(Pro)4-OH was prepared by reacting a proline tetrapeptide with LiOHH2O; the molar ratio of the proline tetrapeptide to LiOHH2O was 1:

1. Then, Boc-(Pro)4-OH was dissolved in dry DCM, and 1-hydroxybenzotriazole was added; dendritic alkoxy ether benzylamine was dissolved in dry DCM, and diisopropylethylamine was added; the mixture was purged and evacuated with high-purity nitrogen at least three times; after reacting in an ice-salt bath for at least 30 min, the reaction solutions were mixed, purged and evacuated with high-purity nitrogen at least three times, and reacted again in an ice-salt bath for at least 30 min; then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the product mixture, and the mixture was allowed to cool naturally to room temperature overnight; the reaction was stopped after TLC to confirm complete reaction, and the mixture was washed at least three times with saturated sodium bicarbonate solution and potassium bisulfate solution (at least 10 wt.%), the organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated by filtration; the intermediate product Et-(Pro)4-Boc was obtained by column chromatography purification, with the following structural formula: Where: n = 4, p = 3, X is OEt; The molar ratio of Boc-(Pro)4-OH, 1-hydroxybenzotriazole, dendritic alkoxy ether moiety, diisopropylethylamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1:2:4:

4. b. The process of imine nucleophilic attack on DASA receptors: The Et-(Pro)4-Boc prepared in step a was dissolved in dry DCM. An ethyl acetate solution of hydrochloric acid was added under ice-salt bath conditions, and the reaction was carried out at a temperature not exceeding -15°C for at least 12 hours. The reaction was stopped after TLC to confirm completion. The ethyl acetate was removed by rotary evaporation, and saturated sodium bicarbonate solution was added dropwise until no more bubbles were generated. Then, a sodium hydroxide solution with a mass percentage concentration of not less than 10 wt.% was added to adjust the pH of the mixture to 10. The mixture was lyophilized to remove water. DCM was added to the resulting solid to dissolve it completely. Anhydrous magnesium sulfate was added for drying. The solvent was evaporated after filtration. The resulting product was dissolved in dry tetrahydrofuran solution, and a DASA acceptor was added. The solution immediately turned deep purple. The reaction was carried out overnight under a dry nitrogen atmosphere at a temperature not lower than 40°C. After the reaction was complete, the solvent was evaporated, and the product was purified by column chromatography to obtain the target product Et-(Pro)4-DASA, whose structural formula is: Where: n = 4, p = 3, X is OEt; the molar ratio of the product obtained using Et-(Pro)4-Boc to the DASA receptor is 1:2.

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