Ligand molecule, nanowire and preparation method and application thereof

Abstract

The invention discloses a ligand molecule, a nanowire as well as a preparation method and application of the nanowire, the ligand molecule is obtained by cross-linking trifluoromethylphenothiazine azide and a Linker molecule, and further, the ligand-protein interaction force of the trifluoromethylphenothiazine azide and calmodulin is utilized to successfully induce the assembly of the protein nanowire. According to the invention, the successful assembly of the nanowire is realized by regulating and controlling the size of the coupling molecule between the double-end ligands, the assembly method of the calmodulin CaM one-dimensional nanowire is expanded, and a new scheme is provided for the assembly of other protein nanowires with ligand molecules.

Description

Technical Field

[0001] This invention relates to a nanomaterial, and more particularly to a ligand molecule, nanowires, their preparation methods, and applications. Background Technology

[0002] Proteins are fundamental to life, participating in all basic life processes. They possess complex functions and precise three-dimensional structures, making them ideal building blocks. Common assembly strategies include supramolecular assembly and chemical cross-linking, with supramolecular assembly being a rapidly developing approach. Supramolecular assembly strategies involve electrostatic interactions, protein-ligand interactions, and metal coordination. Scientists have developed a series of highly complex protein structures, including one-dimensional nanowires, two-dimensional nanosheets, and three-dimensional nanocrystals. Nanowires, in particular, are excellent drug carriers with wide applications in the biomedical field.

[0003] Calmodulin (CaM) is a typical allosteric protein composed of 148 amino acid residues, widely distributed in the human body, and binds to a variety of target enzymes and proteins. Calmodulin exists in three different conformational states that can interconvert. Nanowires constructed based on this protein possess dynamically regulated properties, holding promise for applications in the biological field. However, currently, there is no stable, universal method for synthesizing calmodulin nanowires applicable to biotherapy. Summary of the Invention

[0004] Objectives of the invention: The first objective of this invention is to provide a ligand molecule that can incorporate more functional small molecules; the second objective of this invention is to provide a stable and functional nanowire; and the third objective of this invention is to provide a method for preparing the above-mentioned ligand molecule and nanowire and their applications.

[0005] Technical solution: The ligand molecule of the present invention has the structure of Formula I:

[0006]

[0007] The Linker molecule contains an alkynyl group that reacts with the trifluoromethylphenthiazine azido group.

[0008] Preferably, the ligand molecule has the structure of formula II or III:

[0009]

[0010]

[0011] Where n = 0, 1, 2, 3.

[0012] The nanowires of this invention are prepared by mixing any of the ligand molecules described above with calmodulin (CaM) and calcium ions.

[0013] Preferably, in the nanowire, the molar ratio of calmodulin (CaM) to ligand molecules is 4:1 to 2:1.

[0014] The method for preparing the ligand molecule of the present invention includes the following steps: crosslinking trifluoromethylphenthiazide with a Linker molecule to obtain the ligand molecule.

[0015] Preferably, the crosslinking method is a click reaction.

[0016] Preferably, the molar ratio of the trifluoromethylphenthiazide azidophosphate to the Linker molecule is 1:2 to 1:3.

[0017] The method for preparing nanowires according to the present invention includes the following steps: adding calcium ions and ligand molecules to calmodulin CaM solution, stirring, and obtaining nanowires.

[0018] Preferably, the molar ratio of calmodulin (CaM) to ligand molecules is 1:2 to 1:4; and the molar amount of calcium ions is more than 4 times that of calmodulin (CaM).

[0019] The application of the ligand molecules described in this invention in the preparation of nanowires.

[0020] The application of the nanowires described in this invention in the biomedical field.

[0021] Invention Principle: This invention proposes for the first time the application of trifluoromethylphenothiazine-based bipolar ligand molecules in the preparation of one-dimensional protein nanowires. The synthesis of these bipolar ligands mainly relies on a click chemistry reaction between the azide group of trifluoromethylphenothiazine and the alkyne group on the linker molecule. This ligand molecule drives protein-ligand interactions and protein-protein interactions, inducing the assembly of one-dimensional protein nanowires. Protein-ligand interactions are a type of excellent supramolecular interaction. By utilizing bipolar protein ligands and introducing certain cross-linking molecules, the assembly of protein nanowires can be achieved.

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention synthesizes a small molecule based on trifluoromethylphenthiazide azidophosphate, further enriching the application range of protein nanowires by introducing more functional small molecule ligands. The assembly of protein nanowires is successfully induced by the ligand-protein interaction between trifluoromethylphenthiazide azidophosphate and calmodulin. The nanowires of this invention exhibit good stability and can be further applied in the biomedical field as biological templates. This invention expands the assembly method of one-dimensional calmodulin nanowires, providing a new approach for the assembly of other protein nanowires with ligand molecules. Attached Figure Description

[0023] Figure 1 The hydrogen NMR spectrum (400MHz, CDCl3, 298K) of the OEG3-diPT of this invention is shown.

[0024] Figure 2 The NMR spectrum of the NodiPT of this invention is 1H NMR (400MHz, CDCl3, 298K).

[0025] Figure 3 The NMR spectrum of the N1diPT of this invention is 1H NMR (400MHz, CDCl3, 298K).

[0026] Figure 4 The NMR spectrum of the N2diPT of this invention is 1H NMR (400MHz, CDCl3, 298K).

[0027] Figure 5 The NMR spectrum of the N3diPT of this invention is 1H NMR (400MHz, CDCl3, 298K).

[0028] Figure 6 This is a TEM characterization image of the morphology of the OEG3-diPT system assembly of the present invention.

[0029] Figure 7 This is a TEM characterization image of the morphology of the N0diPT system assembly of the present invention.

[0030] Figure 8 This is a TEM characterization image of the morphology of the N1diPT system assembly of the present invention.

[0031] Figure 9 This is a TEM characterization image of the morphology of the N2diPT system assembly of the present invention.

[0032] Figure 10 This is a TEM characterization image of the morphology of the N3diPT system assembly of the present invention.

[0033] Figure 11 This is the ultraviolet absorption spectrum of the N3diPT of this invention. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] The structural formula of PT-yne in this embodiment of the invention is as follows:

[0036] In the embodiments of the present invention, NDI-di-OEG n The structural formula for -N3 is:

[0037] n = 0, 1, 2 or 3.

[0038] Example 1

[0039] The synthetic ligand molecule OEG3-diPT has the following structural formula:

[0040]

[0041] The specific implementation scheme is as follows: 73.2 mg N3-OEG3-N3 (0.30 mmol), 0.228 g PT-yne (0.75 mmol), and 51.9 mg pentamethyldiethylenetriamine anhydride (0.30 mmol, PMDTA) were dissolved in 5 mL of dry DMF. After purging with argon for 5 minutes, 21.5 mg cuprous bromide (0.15 mmol) was added, and purging with argon continued for 10 minutes. The solvent was evaporated, and the crude product was separated by silica gel column chromatography (eluent: DCM / CH3OH = 100 / 1, av / v), with a yield of 85%. 1 H NMR(δ,ppm,CDCl3):7.47(s,2H,triazole);7.25(m,4H,benzene);7.10(m,4H,benzene);6.94(m,4H,benzene);6.82(m,2H,benzene);5.19(s,4H,-NCH2-) ;4.40(t,4H,-NCH2CH2-triazole); 3.68(t,4H,-NCH2CH2-triazole).3.33(t,4H,-OCH2CH2OCH2CH2-triazole); 3.27(t,4H,-OCH2CH2OCH2CH2-triazole). Figure 1 The hydrogen NMR spectrum of OEG3-diPT (400 MHz, CDCl3, 298 K).

[0042] Example 2

[0043] The synthetic ligand molecule N0diPT has the following structural formula:

[0044]

[0045] The specific implementation scheme is as follows: 0.15 g NDI-di-OEG0-N3 (0.30 mmol), 0.228 g PT-yne (0.75 mmol), and 51.9 mg pentamethyldiethylenetriamine anhydride (0.30 mmol, PMDTA) were dissolved in 5 mL of dry DMF. After purging with argon for 5 minutes, 21.5 mg cuprous bromide (0.15 mmol) was added, and purging with argon was continued for 10 minutes. The solvent was evaporated, and the crude product was separated by silica gel column chromatography (eluent: DCM / CH3OH = 20 / 1, v / v), with a yield of 75%. 1 H NMR(δ,ppm,CDCl3):8.57(m,4H,benzene);7.46(s,2H,triazole);7.09(m,8H,benzene);7.14(m,4H,benzene);6.94(m,2H, benzene); 6.80(m,2H,benzene); 5.14(s,4H,-NCH2-); 4.75(t,4H,-NCH2CH2-triazole); 4.61(t,4H,-NCH2CH2-triazole). Figure 2 The NMR spectrum of N0diPT is 1H NMR (400MHz, CDCl3, 298K).

[0046] Example 3

[0047] The synthetic ligand molecule N1diPT has the following structural formula:

[0048]

[0049] The specific implementation scheme is as follows: 0.17 g NDI-di-OEG1-N3 (0.30 mmol), 0.228 g PT-yne (0.75 mmol), and 51.9 mg pentamethyldiethylenetriamine anhydride (0.30 mmol, PMDTA) were dissolved in 5 mL of dry DMF. After purging with argon for 5 minutes, 21.5 mg cuprous bromide (0.15 mmol) was added, and purging with argon was continued for 10 minutes. The solvent was evaporated, and the crude product was separated by silica gel column chromatography (eluent: DCM / CH3OH = 20 / 1, v / v), with a yield of 75%. 1H NMR(δ,ppm,CDCl3):8.50(m,4H,benzene);7.55(s,2H,triazole);7.09(m,8H,benzene);6.90(m,4H,benzene);6.80(m,2H,benzene);5.1 4(s,4H,-NCH2-); 4.42(t,4H,-OCH2CH2-triazole); 4.21(t,4H,-OCH2CH2N-); 3.79(t,4H,-OCH2CH2N-); 3.65(t,4H,-OCH2CH2-triazole). Figure 3 The NMR spectrum of N1diPT is 1H NMR (400MHz, CDCl3, 298K).

[0050] Example 4

[0051] The synthetic ligand molecule N2diPT has the following structural formula:

[0052]

[0053] The specific implementation scheme is as follows: 0.18 g NDI-di-OEG2-N3 (0.30 mmol), 0.228 g PT-yne (0.75 mmol), and 51.9 mg pentamethyldiethylenetriamine anhydride (0.30 mmol, PMDTA) were dissolved in 5 mL of dry DMF. After purging with argon for 5 minutes, 21.5 mg cuprous bromide (0.15 mmol) was added, and purging with argon continued for 10 minutes. The solvent was evaporated, and the crude product was separated by silica gel column chromatography (eluent: DCM / CH3OH = 20 / 1, v / v), with a yield of 75%. 1 HNMR(δ,ppm,CDCl3):8.63(m,4H,benzene);7.63(s,2H,triazole);7.03(s,10H,benzene);6.81(m,4H,benzene);5.19(s,4H,-NCH2-);4.41(t,4H,-O CH2CH2-triazole); 4.31(t,4H,-OCH2CH2N-); 3.72(m,8H,-OCH2CH2OCH2CH2N-); 3.46(t,4H,-OCH2CH2OCH2CH2N-); 3.41(t,4H,-OCH2CH2-triazole). Figure 4 The hydrogen NMR spectrum of N2diPT (400MHz, CDCl3, 298K).

[0054] Example 5

[0055] The synthetic ligand molecule N3diPT has the following structural formula:

[0056]

[0057] The specific implementation scheme is as follows: 0.20 g NDI-di-OEG3-N3 (0.30 mmol), 0.228 g PT-yne (0.75 mmol), and 51.9 mg pentamethyldiethylenetriamine anhydride (0.30 mmol, PMDTA) were dissolved in 5 mL of dry DMF. After purging with argon for 5 minutes, 21.5 mg cuprous bromide (0.15 mmol) was added, and purging with argon was continued for 10 minutes. The solvent was evaporated, and the crude product was separated by silica gel column chromatography (eluent: DCM / CH3OH = 20 / 1, v / v), with a yield of 75%. 1 H NMR(δ,ppm,CDCl3):8.68(m,4H,benzene);7.54(s,2H,triazole);7.03(s,4H,benzene);6.94(m,4H, benzene); 6.90 (s, 2H, benzene); 6.88 (t, 2H, benzene); 6.80 (d, 2H, benzene); 5.18 (s, 4H, -NCH2-); 4. 46(t,4H,-OCH2CH2-triazole); 4.40(t,4H,-OCH2CH2N-); 3.76(m,8H,-OCH2CH2OCH2CH2N-); 3.59(t,4 H,-OCH2CH2OCH2CH2N-); 3.46(t,4H,-OCH2CH2OCH2CH2-triazole); 3.39(t,2H,-OCH2CH2-triazole). Figure 5 The hydrogen NMR spectrum of N3diPT (400MHz, CDCl3, 298K).

[0058] Example 6

[0059] The assembly morphology of OEG3-diPT was characterized by transmission electron microscopy (TEM), and it was observed that OEG3-diPT was assembled to form one-dimensional nanowires. Figure 6 TEM images show the morphology of the OEG3-diPT system assembly. Experimental results also indicate that in Ca... 2+ In the presence of protein-ligand and protein-protein interactions, the assembly of one-dimensional nanowires is dominated by protein-ligand and protein-protein interactions.

[0060] Example 7

[0061] The morphology of protein assemblies was influenced by adjusting the water solubility and segment flexibility of ligand molecules, and the stability of assemblies under different experimental conditions was evaluated. The specific implementation plan is as follows: A series of small ligand molecules (N0diPT, N1diPT, N2diPT, N3diPT) containing OEG segments of different lengths were synthesized, and the concentration of the assemblies was controlled at 3.0 μM. Ca was added... 2+ Subsequently, the solutions of all four systems became turbid. TEM characterization of the four systems revealed that they could all assemble into one-dimensional nanowires. Figures 7-10 TEM images show the morphology of the N0diPT, N1diPT, N2diPT, and N3diPT system assemblies, respectively.

[0062] The results showed that the length of the OEG chain segment had no significant effect on the assembly behavior. Different ligand molecules could induce the formation of stable and ordered one-dimensional nanowires. The water solubility of the ligand molecules and the flexibility of the chain segments had little effect on the morphology of the assembly, demonstrating the universality of this protein assembly method and the stability of the resulting assembly structure. The assemblies formed by these four systems were all very stable and could remain in solution for more than three months.

[0063] Example 8

[0064] 2 mg / mL of N3diPT molecules from Example 7 was dissolved in DMSO solvent and measured using a UV spectrophotometer. Figure 11 This is the UV absorption spectrum of N3diPT from this invention. Experimental results show that the molecule has an absorption peak in the 400 nm–600 nm range, indicating that it possesses certain photothermal properties. Protein nanowires constructed based on this property hold promise for applications in biomedical engineering. Furthermore, combining the results of Examples 6 and 7, it is evident that there are no other supramolecular forces between naphthalimide molecules; their primary function is merely linkage. This demonstrates that altering the structure of the linker portion of the ligand molecule does not significantly affect the assembly behavior, allowing for greater ligand selectivity. Naphthalimide can be replaced with small molecule ligands with specific functions.

[0065] This invention designed and synthesized a series of biterminated ligand molecules, utilizing CaM allosteric reactions to drive protein-ligand and protein-protein interactions, resulting in the formation of one-dimensional protein nanowires. The application of these novel biterminated ligand molecules in the assembly of calmodulin nanowires was explored. The resulting one-dimensional nanowires exhibited good stability and hold promise as important templates for biological research. Furthermore, different ligand molecules were able to guide proteins to form stable and ordered assembly structures, validating the broad applicability of the assembly method. Future research will introduce more functional small molecule ligands for assembly to expand the application areas of protein assemblies.

Claims

1. A ligand molecule, characterized in that, It has the structure of Formula I: The Linker molecule contains an alkynyl group that reacts with the trifluoromethylphenthiazine azido group.

2. The ligand molecule according to claim 1, characterized in that, The ligand molecule has a structure of formula II or III: Where n = 0, 1, 2, 3.

3. A nanowire, characterized in that, It is prepared by mixing the ligand molecule described in any one of claims 1-2 with calmodulin CaM and calcium ions.

4. The nanowire according to claim 3, characterized in that, In the nanowires, the molar ratio of calmodulin (CaM) to ligand molecules is 4:1-2:

1.

5. A method for preparing the ligand molecule according to claim 1, characterized in that, The process includes the following steps: cross-linking trifluoromethylphenthiazide azidophosphate with Linker molecules to obtain ligand molecules.

6. The preparation method according to claim 5, characterized in that, The crosslinking method is a click reaction.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the azidotrifluoromethylphenthiazide to the Linker molecule is 1:2-1:

3.

8. A method for preparing the nanowires according to claim 3, characterized in that, Includes the following steps: Calcium ions and ligand molecules were added to a calmodulin (CaM) solution, and the mixture was stirred to obtain nanowires.

9. The use of a ligand molecule according to any one of claims 1-2 or a ligand molecule prepared by any one of claims 5-7 in the preparation of nanowires.

10. The application of a nanowire according to any one of claims 3-4 or a nanowire prepared by the method of claim 8 in the biomedical field.

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