Design method of probe CazFPC-SNAP and its visualization detection of protein aggregates

Abstract

The present invention discloses a design method of a novel probe CazFPC-SNAP and its visualization detection of protein aggregates. The CazFPC-SNAP is synthesized by a three-step method of conjugating phenylboronic acid pinacol ester with a 4-hydroxybenzylidene imidazolinone core and an anchoring biotag SNAP. Phenylboronic acid pinacol ester serves as a hydrogen peroxide recognition site, and the introduced biotag SNAP is used to identify and label the target protein. The probe CazFPC-SNAP can not only be used to quickly detect the concentration level of hydrogen peroxide, but also can realize the visualization imaging of protein misfolding and aggregation in living cells. It has an obvious fluorescent response to the viscosity change in the protein folding system and can identify protein aggregates with the naked eye. It has low cytotoxicity, good biocompatibility, and stable fluorescent properties. It has extremely important applications in the fields of bioimaging, fluorescent labeling, biosensors, and drug development. The present invention solves the problem that insoluble misfolded proteins in cells are difficult to detect.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent compounds and their preparation and application, involving the fields of organic synthesis, chemical quantitative detection and analysis, cell biology and protein folding omics; in particular, it relates to a design method of a probe CazFPC-SNAP and its visual detection of protein aggregates; specifically, it relates to the design and synthesis of a 4-hydroxybenzylidene imidazolinone hydrogen peroxide probe CazFPC-SNAP and its visual detection of protein aggregates. Background Art

[0002] With the increasing aging of the population, the incidence of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) is increasing in the elderly. These diseases are associated with the degeneration and death of specific neurons and the formation of intraneuronal inclusion bodies caused by the aggregation of mutant proteins. It is generally believed that the aggregation of soluble proteins into oligomers and ultimately into insoluble fibrils is a key pathogenic mechanism of neurotoxicity, and superoxide dismutase (SOD1) has been implicated in these neurodegenerative diseases. Studies have shown that excessive levels of oxidative factors can cause proteins to lose their original function, leading to misfolding and aggregation, ultimately leading to the development of neurological diseases. As proteins undergo a stimulated transition from their normal structure to a folded, aggregated state, their viscosity gradually increases. Based on these physiological characteristics, a number of fluorescence imaging tools based on viscosity change detection are used to detect protein aggregation for disease diagnosis. Therefore, the detection of hydrogen peroxide concentration levels and the visualization of protein aggregation processes in biological samples are essential and have important implications for the monitoring and diagnosis of neurodegenerative diseases. This technology uses SOD1 as the research object. Through fluorescent probe technology, it uses biomimetic chromophores as fluorescence imaging tools to perform fluorescence imaging in living cells, achieving the purpose of visually identifying neurodegenerative pathogenic proteins. This fills the gap in the rapid detection of protein aggregation by fluorescence methods and is expected to serve as an effective drug screening platform.

[0003] The 4-hydroxybenzylidene imidazolinone fluorescent chromophore (HBI) is the core structure of green fluorescent protein (GFP) luminescence. Currently, researchers have developed a variety of methods for the chemical synthesis of GFP and its analogs. The HBI fluorescent chromophore has excellent properties such as high fluorescence quantum yield, large molar absorption coefficient, narrow fluorescence spectrum peak, high sensitivity, and good photostability. Its application in fluorescence imaging and sensing, optical devices and other fields is increasingly attracting attention. Through the rich functional modification of HBI fluorophores, their absorption and emission can even be red-shifted to the red / near-infrared (NIR) region. Conjugated hydrogen peroxide recognition sites can sensitively detect hydrogen peroxide levels. In addition, the introduction of a specific protein tag SNAP-linker can not only detect misfolded and aggregated proteins, but also detect changes in the viscosity of the intracellular environment, which has great biological value. Summary of the Invention

[0004] Purpose of the invention: The first technical problem solved by the present invention is to provide an effective fluorescent sensor for detecting hydrogen peroxide concentration levels in vitro.

[0005] The second technical problem to be solved by the present invention is to provide an optimized chemical preparation method of 4-hydroxybenzylidene imidazolidinone chromophore, which greatly improves the yield.

[0006] The third technical problem to be solved by the present invention is to provide a small molecule fluorescent probe capable of detecting the viscosity of the intracellular microenvironment.

[0007] The fourth technical problem to be solved by the present invention is to provide a small molecule fluorescent probe for detecting soluble and insoluble SOD1-A4V-SNAP proteins.

[0008] Technical Solution: The probe CazFPC-SNAP described in the present invention contains a phenylboronic acid pinacol ester structure and a protein recognition tag SNAP-linker, which are used to recognize hydrogen peroxide and connect with the target protein through bioorthogonal reactions, thereby achieving visual detection of protein misfolding and aggregation.

[0009] The chemical structure of CazFPC-SNAP is shown in formula (I):

[0010]

[0011] Furthermore, the preparation method of the novel probe CazFPC-SNAP is as follows: first, a dimethylamino group is modified on HBI; secondly, phenylboronic acid pinacol ester is used to condense to a fluorescent core by zinc chloride catalysis, and finally, the Boc protecting group is removed using trifluoroacetic acid and then added to DMF simultaneously with the SNAP-linker without further purification. HOBt·H2O is added, followed by triethylamine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), and the reaction mixture is stirred overnight. After the reaction is completed, the solution is quenched with water, extracted with DCM, and the organic portion is collected, dried over anhydrous Na2SO4, and then vacuum dried to remove the solvent. The compound is further purified by thin layer chromatography to obtain the probe CazFPC-SNAP as a red solid powder;

[0012] Specifically, step (1) modifying HBI with a dimethylamino group: 1 equivalent of glycine tert-butyl ester hydrochloride and 1.1 equivalents of ethyl ethylimidate hydrochloride were mixed with 1 equivalent of NaOH in 20 mL of EtOH, and stirred at room temperature for 1 hour, followed by addition of 1 equivalent of 4-(dimethylamino)benzaldehyde, and stirring at 25° C. for 12 hours before termination of the reaction; compound 1 was purified by thin layer chromatography (PE:EA=2:1) to obtain an orange solid;

[0013] Step (2), adding 1 equivalent of the purified compound 1 and 2 equivalents of phenylboronic acid pinacol in a pressure tube, adding 10 mL of Dioxane and 0.01 mmol of catalyst zinc chloride, and raising the temperature to 100 ° C. to react for 12 hours, and then purifying by thin layer chromatography (PE: EA = 1: 1) to obtain a red solid compound 2; dissolving 1 equivalent of compound 2 in DCM, adding 2 equivalents of trifluoroacetic acid, stirring at 25 ° C. for 2 hours, and then purifying by thin layer chromatography (DCM: EtOH = 10: 1) to obtain a red solid compound 3;

[0014] Step (3): 5 equivalents of lithium aluminum tetrahydride were dissolved in 150 mL of THF, 1 equivalent of 4-cyanobenzaldehyde was added, and the mixture was allowed to react for 30 minutes, then heated to 85°C and reacted for 20 hours. After the reaction was completed, 10 mL of water and 4 mL of 20% NaOH were added under an ice bath to quench the reaction; the mixture was filtered through celite, the filtrate was collected, and 45 mL of 1 M HCl was added. The mixture was allowed to stand for 10 minutes, and then extracted with EA. The aqueous phase was collected and freeze-dried to obtain compound 4 as a white solid.

[0015] Step (4), 1 equivalent of compound 4 and 2 equivalents of ethyl trifluoroacetate were dissolved in 20 mL of MeOH. After 10 min, 1 equivalent of triethylamine was added and the mixture was stirred at room temperature for 12 h. The mixture was purified by thin layer chromatography (PE:EA=1:1) to obtain compound 5 as a white solid.

[0016] Step (5), adding 1 equivalent of 2-amino-6-chloropurine and 2 equivalents of 1-methylpyrrolidine to 20 mL of DMF, stirring at room temperature for 48 h; collecting the solid by filtration, washing the solid with 100 mL of ether, and drying in vacuo to obtain compound 6;

[0017] Step (6): 1 equivalent of compound 5 and 2 equivalents of compound 6 were dissolved in 10 mL of DMF, 10 equivalents of potassium tert-butoxide were added, and stirred for 12 h. The mixture was purified by thin layer chromatography (DCM:EtOH=10:1) to obtain compound 7 as a white solid;

[0018] Step (7), 1 equivalent of compound 7 was dissolved in 10 mL of a mixture of 80% methanol and 20% water, 5 equivalents of potassium carbonate was added, and the mixture was refluxed at 80°C for 6 h. The mixture was then purified by thin-layer chromatography (DCM:MeOH=10:1) to obtain a white solid SNAP-linker;

[0019] Step (8): In 10 mL of DMF, 1 equivalent of compound 3, 1.2 equivalents of SNAP-linker, 1.2 equivalents of HOBt.H2O, 4 equivalents of triethylamine, and 1.5 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added; after reacting for 12 h, the mixture was purified by thin-layer chromatography (DCM:EtOH = 10:1) to obtain CazFPC-SNAP, a red powder solid.

[0020] Furthermore, the fluorescent probe molecule CazFPC-SNAP is used to rapidly detect hydrogen peroxide concentration levels in vitro.

[0021] Furthermore, the fluorescent probe molecule CazFPC-SNAP is used to detect SOD1-A4V-SNAP protein misfolding in vitro.

[0022] Furthermore, the fluorescent probe molecule CazFPC-SNAP is used in the detection of viscosity during changes in the intracellular microenvironment.

[0023] Furthermore, the fluorescent probe molecule CazFPC-SNAP is used to detect the insoluble protein SOD1-A4V-SNAP in living cells.

[0024] The present invention discloses a bifunctional fluorescent probe CazFPC-SNAP based on a 4-hydroxybenzylidene imidazolinone (HBI) core, which can be used for hydrogen peroxide detection and visual detection of protein misfolding. The present invention also discloses a method for preparing the 4-hydroxybenzylidene imidazolinone small molecule probe CazFPC-SNAP and its detection performance for hydrogen peroxide. CazFPC-SNAP is synthesized by a three-step method of conjugating a 4-hydroxybenzylidene imidazolinone core to phenylboronic acid pinacol ester and an anchoring biotag SNAP. Phenylboronic acid pinacol ester serves as a hydrogen peroxide recognition site, and the introduced biotag SNAP is used to identify and label the target protein. This probe CazFPC-SNAP is a fluorescence-on probe that can not only be used to rapidly detect hydrogen peroxide concentration levels, but also to achieve visual imaging of protein misfolding and aggregation in living cells. It has a significant fluorescence response to viscosity changes in the protein folding system, allowing the naked eye to identify protein aggregates. Because the small molecule probe CazFPC-SNAP is designed based on the 4-hydroxybenzylidene imidazolinone luminescent core, it exhibits low cytotoxicity, good biocompatibility, and stable fluorescence properties. Its large molar absorption coefficient and strong resistance to background interference make it an excellent probe design platform with crucial applications in bioimaging, fluorescent labeling, biosensors, and drug development. The fluorescent probe disclosed in this invention provides a simple fluorescent tool for detecting hydrogen peroxide, while also addressing the difficulty in detecting insoluble misfolded proteins within cells.

[0025] Beneficial effects: Compared with the prior art, the present invention is characterized in that: the yield of the synthesis method disclosed in the present invention is greatly improved, which is beneficial to subsequent research and synthesis work; the structure, synthesis, fluorescence properties and application of the 4-hydroxybenzylidene imidazolinone analog probe CazFPC-SNAP in the present invention have not been reported in existing literature; through the optimized synthesis method, the synthesis yield of 4-hydroxybenzylidene imidazolinone is improved, and the key element for hydrogen peroxide recognition (phenylboronic acid pinacol group) is conjugated to the diethylamino structure to ensure high viscosity response, and the dual-function fluorescence sensor CazFPC-SNAP is designed and synthesized; it can be used not only to detect the concentration of hydrogen peroxide, but also to detect the concentration of hydrogen peroxide. At the same time, it can realize the visualization imaging of protein misfolding and aggregation in living cells; the CazFPC-SNAP probe is a fluorescence "enhanced" probe, which maintains a significant fluorescence enhancement response in the presence of hydrogen peroxide; the probe exhibits weak fluorescence in the normal proteome, but emits a bright fluorescence enhancement effect in high-viscosity insoluble protein aggregates, realizing the naked-eye visualization detection of fluorescence from "no" to "yes"; the probe maintains a highly sensitive fluorescence response to hydrogen peroxide, has high fluorescence quantum yield, large molar absorption coefficient, high signal-to-noise ratio, good biocompatibility and other excellent performance, and has extremely important application value in the fields of living cell imaging, fluorescence sensors, and biofluorescent labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the synthetic route diagram of the present invention;

[0027] Figure 2 3. This is a comparison of the fluorescence responses of the probe CazFPC-SNAP (10 μM) at different hydrogen peroxide concentration levels according to an embodiment of the present invention;

[0028] Figure 3 This is a comparison diagram of the temperature-induced in vitro purified SOD1-A4V-SNAP protein and the SNAP-Tag protein coupled with the probe CazFPC-SNAP in the examples of the present invention;

[0029] Figure 4 Schematic diagram of an experiment using the probe CazFPC-SNAP to detect insoluble aggregation of SOD1-A4V-SNAP protein induced by hydrogen peroxide in HEK293 cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings:

[0031] Example 1 Preparation of probe molecule CazFPC-SNAP

[0032] Synthesis of intermediate compound 1

[0033] 1 equivalent of glycine tert-butyl ester hydrochloride and 1.1 equivalents of ethyl acetimidate hydrochloride were mixed with 1 equivalent of NaOH in EtOH and stirred at room temperature for 1 hour; 1 equivalent of 4-(dimethylamino)benzaldehyde was added and stirred at 25°C overnight; the reaction was stirred at 25°C overnight and then quenched with water (5 mL) and extracted with diethyl ether (3×10 mL); the organic portion was collected and the diethyl ether was removed by evaporation under reduced pressure; the compound was further purified by thin layer chromatography (PE:EA=2:1) and finally dried in vacuo to obtain compound 1 as an orange solid.

[0034] Synthesis of intermediate compounds 2 and 3

[0035] After 1 equivalent of the purified compound 1 and 2 equivalents of phenylboronic acid pinacol were added to 10 mL of Dioxane in a pressure tube and fully dissolved, 0.01 mmol of the catalyst zinc chloride was added and the temperature was raised to 100 ° C for overnight reaction. After 12 hours, the reaction progress was monitored by TLC. After the reaction was completed, it was extracted three times with water and DCM. The organic phase was collected and the DCM was removed by evaporation under reduced pressure. It was purified by thin layer chromatography (PE:EA=1:1) to obtain a red solid compound 2. 1 equivalent of compound 2 was dissolved in DCM, 2 equivalents of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. The reaction was terminated with water and the reaction solution was extracted with DCM. The organic phase was collected and the DCM was removed by evaporation under reduced pressure. It was purified by thin layer chromatography (DCM:EtOH=10:1) to obtain a red solid compound 3.

[0036] Synthesis of intermediate compound 4

[0037] Under ice-cooling, 5 equivalents of lithium aluminum tetrahydride were dissolved in 150 mL of THF, and 1 equivalent of 4-cyanobenzaldehyde dissolved in 30 mL of THF was added dropwise. The mixture was mixed by rotation at room temperature for 30 minutes and then incubated in an 85°C oil bath for 20 hours. After the reaction, 10 mL of water and 4 mL of 20% NaOH were added under ice-cooling to quench the reaction. The filtrate was collected by filtration through celite, and the solid was washed several times with THF. The THF was removed by evaporation under reduced pressure, and 45 mL of 1 M HCl was added. The mixture was allowed to stand for a while to form a salt, which was then extracted with EA. The aqueous phase was collected and lyophilized (white solid). The solid was washed with acetone and evaporated under reduced pressure to obtain compound 4 as a white solid.

[0038] Synthesis of intermediate compound 5

[0039] Under ice bath conditions, 1 equivalent of compound 4 and 2 equivalents of ethyl trifluoroacetate were mixed in MeOH. After 10 minutes, 1 equivalent of triethylamine was added. The ice bath was removed and the mixture was stirred at room temperature overnight. Thin layer chromatography (PE:EA=1:1) was used for purification to obtain compound 5 as a white solid.

[0040] Synthesis of intermediate compound 6

[0041] Dissolve 1 equivalent of 2-amino-6-chloropurine and 2 equivalents of 1-methylpyrrolidine in DMF and stir at room temperature for 48 hours. After the reaction, filter the solid and place it in a beaker. Add 20 mL of acetone to the precipitate, stir, let it stand, and filter. Rinse with 100 mL of ether and dry under vacuum to obtain compound 6 without further purification.

[0042] Synthesis of intermediate compound 7

[0043] One equivalent of the purified compound 5 and two equivalents of compound 6 were dissolved in DMF, and an excess of potassium tert-butoxide was added and stirred for 12 h. The reaction was monitored by TLC. DCM was removed by evaporation under reduced pressure, and the mixture was purified by thin layer chromatography (DCM:EtOH=10:1) to obtain compound 7 as a white solid.

[0044] Synthesis of the intermediate compound SNAP-linker

[0045] The purified compound 7 was dissolved in a mixture of 80% methanol and 20% water. Excess potassium carbonate was added and the mixture was refluxed at 80°C for 6 h. The reaction was monitored by TLC, the solvent was removed by evaporation under reduced pressure, and the mixture was purified by thin-layer chromatography (DCM:MeOH=10:1) to obtain a white solid SNAP-linker.

[0046] Synthesis of target compound probe CazFPC-SNAP

[0047] In DMF, 1 equivalent of compound 3, 1.2 equivalents of SNAP-linker, 1.2 equivalents of HOBt . H2O, 4 equivalents of triethylamine, and 1.5 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and stirred overnight, then quenched with water and extracted with DCM. The organic fraction was collected, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The compound was further purified by thin-layer chromatography (DCM:EtOH = 10:1) to obtain the probe CazFPC-SNAP as a red solid.

[0048] Analysis and characterization of the probe CazFPC-SNAP:

[0049] H NMR 1H NMR (600MHz, DMSO-d6) δ12.45(s,1H),8.78(t,J=5.4Hz,1H),8.22(d,J=8.1Hz,2H),7.9 2(d,J=15.7Hz,1H),7.82(s,1H),7.79(d,J=7.9Hz,2H),7.75(d,J=7.8Hz,2H),7.38(d,J =7.8Hz,2H),7.29(d,J=7.8Hz,2H),7.21(d,J=15.8Hz,1H),7.00(s,1H),6.81(d,J=8.8 Hz,2H),6.29(s,2H),5.42(s,2H),4.57(s,2H),4.32(s,2H),3.05(s,6H),1.29(s,12H).

[0050] C NMR 13 C NMR(151MHz,DMSO)δ170.03,167.51,160.08,156.94,139.36,138.59,138.02,135.34, 134.86,128.97,127.83,127.78,122.54,116.20,112.29,84.28,42.59,25.43,25.12.

[0051] High-resolution mass spectrometry HRMS: Calculated: [M+H] + =754.3558,Obsd,754.3566

[0052] Example 2 Relative fluorescence intensity of the probe CazFPC-SNAP (10 μM) at different hydrogen peroxide concentration levels (0-100 μM)

[0053] The fluorescence response of the probe CazFPC-SNAP to different concentrations of hydrogen peroxide was measured; the changes in the probe fluorescence intensity were observed at different hydrogen peroxide concentration gradients, such as 0μM, 10μM, 20μM, 30μM, 50μM, 80μM, and 100μM; the CazFPC-SNAP probe concentration was maintained at 10μM, and hydrogen peroxide solutions of the above concentrations were prepared using PBS buffer, and fluorescence was recorded on a Tecan infiniteM1000Pro fluorescence microplate reader; the fluorescence intensity of the probe fluorescence in 100μM hydrogen peroxide was used for normalization calculations; all fluorescence intensities were divided by the fluorescence intensity in 100μM hydrogen peroxide and the results were plotted as a bar graph; it can be clearly seen from the data characteristics that as the hydrogen peroxide concentration increases, the relative fluorescence intensity of the probe molecule also gradually increases, indicating that the probe molecule CazFPC-SNAP has a rapid detection function for hydrogen peroxide.

[0054] Example 3 Temperature-induced experiments on purified SOD1-A4V-SNAP protein and SNAP-Tag protein coupled with probe CazFPC-SNAP

[0055] This experiment used a temperature gradient of 25 to 59°C (25, 37, 41.8, 45.6, 50.7, 54.5, 57.2, 59°C) to induce protein misfolding and aggregation; each sample contained 42 μM protein and 10 μM probe for binding;

[0056] The buffer system mainly consisted of: 83mM metal ion chelator EDTA, 100mM NaCl, 50mM Tris-HCl, pH 7.5); each sample was incubated at different temperatures for 10 minutes; fluorescence intensity was recorded on a Tecan infiniteM1000Pro fluorescence microplate reader with an excitation wavelength of 500nm; all fluorescence intensities were normalized by the fluorescence intensity of CazFPC-SNAP co-incubated with SOD1-A4V-SNAP at 59°C and plotted as a bar graph; these data indicate that as the temperature increases, the degree of protein aggregation increases and the fluorescence of CazFPC-SNAP increases; these experimental results indicate that the probe CazFPC-SNAP can be used as an excellent fluorescent sensor for protein detection.

[0057] Example 4: Detection of insoluble aggregation of SOD1-A4V-SNAP protein induced by hydrogen peroxide in HEK293 cells using the probe CazFPC-SNAP

[0058] The present invention conducts a cell imaging experiment on the probe CazFPC-SNAP, using human embryonic kidney HEK293 cells. The probe and SOD1-A4V-SNAP plasmid are transfected into the cells and incubated together to allow full protein expression for 24 hours. The experimental group is then added with a 200 μM inducer to induce protein aggregation within the cells. Before imaging, the nuclear dye Hoechst 33342 is used for 30 minutes, and excess probe and dye are rinsed off. The experimental results show that the probe CazFPC-SNAP maintains bright fluorescence in the SOD1-A4V-SNAP aggregation area, demonstrating that the probe can effectively detect insoluble aggregated proteins within living cells.

[0059] The example compound probe CazFPC-SNAP of the present invention has excellent fluorescent properties. It can not only be used to detect the concentration level of hydrogen peroxide, but also realize the visualization imaging of protein misfolding and aggregation in living cells. Its excellent biocompatibility gives it good application prospects in the fields of fluorescent sensors, bioluminescence analysis, fluorescent labeling, etc.

[0060] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The probe CazFPC-SNAP is characterized by: The chemical structure of the probe CazFPC-SNAP is shown in formula (I):

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