Double-response fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a dual-response fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of small molecular probes. The invention provides a chemical structure, a preparation method and application of the double-response fluorescent probe. The background fluorescence of the fluorescent probe P2 prepared by the invention can be ignored. If the probe is located in a high-viscosity medium, near-infrared fluorescence can be emitted at 819nm, so that viscosity detection is realized; when peroxynitrite exists in the system, the conjugated structure of the probe is damaged, and the probe emits green fluorescence at 511nm, so that the recognition of peroxynitrite is achieved. Cell experiments find that the probe can be used for fluorescence imaging of intracellular viscosity and peroxynitrite. The probe can also be used for viscosity imaging analysis of inflammation and diabetic mice. In addition, we can simultaneously monitor changes in viscosity and peroxynitrite levels during ferroptosis.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecule probes, and more particularly to a dual-response fluorescent probe and a preparation method and application thereof. Background Art

[0002] Mitochondria are the "powerhouse" of many eukaryotic cells and play an important role in biological processes such as cell division, autophagy, apoptosis, and cell differentiation. Viscosity, as a key parameter of the mitochondrial microenvironment, can affect a variety of physiological processes, such as intracellular material transport, signal transduction, and intermolecular interactions. In addition, abnormal intracellular viscosity is associated with certain diseases, including diabetes, atherosclerosis, Parkinson's disease, and Alzheimer's disease. Therefore, real-time monitoring of mitochondrial viscosity is of great significance.

[0003] Peroxynitrite is an important reactive oxygen species with the characteristics of strong oxidizing property, strong nucleophilicity and short half-life. Peroxynitrite plays an important role in maintaining redox homeostasis, resisting invasion of foreign bacteria, signal transduction and tumorigenesis. However, excessive peroxynitrite can react irreversibly with biological molecules (such as proteins, nucleic acids, lipids), leading to the loss of function of important components in cells, thereby inducing the occurrence of diseases. Therefore, the detection of peroxynitrite in biological samples is very necessary.

[0004] In the detection of analytes in vivo, fluorescent probes have attracted great attention due to their advantages such as simple synthesis, high sensitivity, and good specificity. To this end, researchers have developed many fluorescent probes that can identify viscosity and peroxynitrite. Although these probes show excellent performance, they are only suitable for the separate determination of viscosity or peroxynitrite, and cannot achieve the simultaneous detection of the two. More importantly, studies have shown that the level of intracellular reactive oxygen species may be correlated with viscosity. Therefore, how to develop a dual-response fluorescent probe that can simultaneously detect viscosity and peroxynitrite is an urgent problem that technicians in this field need to solve. Summary of the invention

[0005] In view of this, the present invention provides a dual-response fluorescent probe for simultaneously detecting viscosity and peroxynitrite, and a preparation method and application thereof.

[0006] Dual-response fluorescent probes are more advantageous for simultaneous detection of viscosity and peroxynitrite. The two fluorescent channels are far apart and have the advantage of being free from fluorescence crosstalk. This dual-function detection is almost unaffected by fluorescence crosstalk, which can greatly reduce the probability of false positive results and improve analytical accuracy.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A dual-response fluorescent probe for simultaneous detection of viscosity and peroxynitrite, the chemical structure of which is as follows:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned dual-response fluorescent probe for simultaneously detecting viscosity and peroxynitrite, comprising the following steps:

[0011] (1) 8-Hydroxyjulolidine and phthalic anhydride were dissolved in toluene, heated to reflux, the solvent was removed, and the compound 3 was obtained by chromatography;

[0012] (2) After the reaction of compound 3 and cyclohexanone in concentrated sulfuric acid, the mixture was poured into crushed ice and stirred, perchloric acid was added and stirred, and the mixture was allowed to stand and then filtered to obtain compound 4;

[0013] (3) Compound 4 and 9-formyl julolidine were dissolved in acetic anhydride, heated to reflux, the solvent was removed, and the fluorescent probe P2 was obtained by chromatography;

[0014]

[0015] Preferably: in step (1), the molar ratio of 8-hydroxyjulolidine to phthalic anhydride is 1:1-2; the reaction temperature is controlled to be 80-100°C, and the reaction is carried out for 8-24 hours; chromatography: using methanol and dichloromethane in a volume ratio of 1:20-50 as eluents, and using silica gel column chromatography.

[0016] Preferably: in step (2), the molar ratio of cyclohexanone, compound 3 and concentrated sulfuric acid is 1:1-2:30-100; the reaction temperature of compound 3 and cyclohexanone is 60-90°C, and the reaction time is 1-3h; the mass ratio of concentrated sulfuric acid to crushed ice is 1:10-30, the control temperature is 0°C, and the stirring time is 2-10min; the mass fraction of perchloric acid is 50%-70%, the molar ratio of perchloric acid to compound 3 is 1:5-10, and the stirring time is 10-20min; the standing time is 1-3h.

[0017] Preferably: the molar ratio of compound 4 to 9-aldehyde julolidine in step (3) is 1:1-2; the reaction temperature is 80°C-100°C, and the reaction time is 2-8h; the chromatography: using methanol and dichloromethane in a volume ratio of 1:20-80 as eluents, and using silica gel column chromatography.

[0018] The present invention also provides the above dual-response fluorescent probe for qualitative or quantitative detection of ONOO - It is used as a detection reagent for ions and / or viscosity.

[0019] The present invention also provides the use of the dual-response fluorescent probe in preparing a peroxynitrite and / or viscosity fluorescent imaging agent in living cells.

[0020] The present invention also provides the use of the dual-response fluorescent probe in preparing a fluorescent imaging agent in the process of cell ferroptosis.

[0021] It can be known from the above technical scheme that compared with the prior art, the present invention discloses a dual-response fluorescent probe and application for simultaneously detecting viscosity and peroxynitrite, and the technical effect obtained is that the fluorescent probe P2 prepared by the present invention, in a low-viscosity phosphate buffer, because the carbon-carbon single bond can rotate freely, the probe itself does not fluoresce; when in a high viscosity environment (such as glycerol), the single bond rotation is limited, and the probe can emit near-infrared fluorescence at 819nm, thereby realizing the detection of viscosity. On the other hand, in the absence of peroxynitrite, the background fluorescence of the probe itself can be ignored; and after reacting with peroxynitrite, the conjugated structure of the probe is destroyed, and green fluorescence will be emitted at 511nm, achieving the identification of peroxynitrite. The probe can be used for fluorescence imaging of viscosity and peroxynitrite in biological samples. In addition, the probe can also be used to monitor the changes in viscosity and peroxynitrite levels during ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The probe P2 provided by the present invention responds to viscosity, wherein: Figure 1 A is the fluorescence emission spectra of the probe in glycerol-phosphate buffer with different ratios; Figure 1 B is the linear relationship between the logarithm of the fluorescence intensity of the probe at 819 nm and the logarithm of the viscosity.

[0023] Figure 2 The probe P2 provided by the present invention responds to peroxynitrite, wherein: Figure 2 A is the fluorescence emission spectra of the probe in peroxynitrite solutions with different concentrations; Figure 2 B is the linear relationship between the fluorescence intensity of the probe at 511 nm and the concentration of peroxynitrite.

[0024] Figure 3 A schematic diagram of the selectivity of the probe P2 provided by the present invention, wherein: Figure 3 The vertical axis is the fluorescence intensity of the probe at 819 nm, and the order of the substances on the horizontal axis is 1.CO3 2- (100μM); 2.SO4 2- (100μM); 3.S 2- (100μM); 4.S2O3 2- (100μM); 5.SO3 2-(100μM); 6.Cl-(100μM); 7.HS-(100μM); 8.HSO3 - (100μM); 9. NO3 - (100μM); 10.Ag + (100μM); 11.Al 3+ (100μM); 12.Ca 2+ (100μM); 13.Cu 2+ (100μM); 14.Fe 2+ (100μM); 15.K + (100μM); 16.Na + (100μM); 17.Mg 2+ (100 μM); 18. Zn 2+ (100μM); 19. Alanine (1mM); 20. Arginine (1mM); 21. Cysteine ​​(1mM); 22. Glutamate (1mM); 23. Homocysteine ​​(1mM); 24. Histidine (1mM); 25. Serine (1mM); 26. H2O2 (100μM); 27. ClO - (100μM); 28.t-BuOOH (100μM); 29.·OH (100μM); 30.t-BuO·(100μM); 31. 1 O2(100μM); 32.NO(100μM); 33.NO2 - (100 μM); 34. C2H5SH (100 μM); 35. Bovine serum albumin (10 μg / mL); 36. Human serum albumin (10 μg / mL); 37. Glycerol. Figure 3 The vertical axis of B is the fluorescence intensity of the probe at 511nm, and the order of the substances on the horizontal axis is 1.CO3 2- (100μM); 2.SO4 2- (100μM); 3.S 2- (100μM); 4.S2O3 2- (100μM); 5.SO3 2- (100μM); 6.Cl-(100μM); 7.HS-(100μM); 8.NO3 - (100μM); 9.Na + (100μM); 10.K + (100μM); 11.Mg 2+ (100μM); 12.Ag + (100μM); 13.Al 3+ (100μM); 14.Ca 2+(100μM); 15.Cu 2+ (100μM); 16.Fe 2+ (100μM); 17.Zn 2+ (100μM); 18. Alanine (1mM); 19. Arginine (1mM); 20. Cysteine ​​(1mM); 21. Glutamic acid (1mM); 22. Homocysteine ​​(1mM); 23. Histidine (1mM); 24. Serine (1mM); 25. C2H5SH (100μM); 26. H2O2 (100μM); 27. ClO - (100μM); 28.t-BuOOH (100μM); 29.·OH (100μM); 30.t-BuO· (100μM); 31.Blank; 32. 1 O2(100μM); 33.NO(100μM); 34.NO2 - (100 μM); 35. Peroxynitrite (30 μM).

[0025] Figure 4 This is a confocal imaging diagram of the probe P2 provided by the present invention in SK-OV-3 cells under different viscosity conditions, wherein: Figure 4 In A, the red channel row is the fluorescent image of the probe in the cell, and the bright field row is the bright field photo of the cell; Figure 4 B is a statistical graph of cell fluorescence intensity.

[0026] Figure 5 This is a confocal imaging diagram of the probe P2 provided by the present invention in HeLa cells after stimulation by exogenous and endogenous drugs, wherein: Figure 5 The green row in A is the peroxynitrite fluorescence image of the probe in the cell, and the bright field row is the bright field photo of the cell; Figure 5 B is a statistical graph of cell fluorescence intensity.

[0027] Figure 6 The probe P2 provided by the present invention is used for imaging of cell ferroptosis, wherein: Figure 6 In A, the control row is the image of the probe in cells without any treatment (control group), and the erastin row is the image of cells treated with erastin; Figure 6 B is a statistical graph of cell fluorescence intensity. DETAILED DESCRIPTION

[0028] Embodiments of the present invention are described below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention.

[0029] Example 1

[0030] A specific synthesis method of a dual-functional fluorescent probe for simultaneous detection of viscosity and peroxynitrite

[0031] 1.89 g 8-hydroxyjulolidine (10 mmol) and 1.48 g phthalic anhydride (10 mmol) were dissolved in 35 mL toluene, and the mixture was heated to 100 ° C. and refluxed for 16 h. After the reaction was completed, the solvent was removed under reduced pressure, and then purified by silica gel column chromatography using methanol and dichloromethane in a volume ratio of 1:20 as eluent to obtain compound 3 (yellow powder, 2.36 g, yield 70%).

[0032]

[0033] 10 mL of concentrated sulfuric acid was cooled to 0°C, and then 1.04 mL of cyclohexanone (10 mmol) and 1.69 g of compound 3 (5 mmol) were added to the concentrated sulfuric acid in sequence, and the reaction solution was heated to 90°C and stirred for 1.5 h. The reaction mixture was then poured into 100 g of ice and stirred for 2 min, and then 1 mL of 70% perchloric acid was added dropwise and stirred continuously for 20 min. After standing for 1 h, the precipitate was collected by filtration to obtain compound 4 (red solid, 1 g, yield 40%).

[0034]

[0035] 500 mg of compound 4 (1 mmol) and 241 mg of 9-formyl julolidine (1.2 mmol) were dissolved in 15 mL of acetic anhydride, and the mixture was refluxed at 100 ° C for 4 h. After the reaction was completed, the solvent was removed under reduced pressure, and then the fluorescent probe P2 (blue powder, 410 mg, yield 60%) was purified by silica gel column chromatography using methanol and dichloromethane in a volume ratio of 1:20 as eluent. 1 H-NMR (400MHz, DMSO-d6) δ13.16(s,1H),8.17(d,J=7.6Hz,1H),7.83(dd,J=7.0Hz,1H ),7.78-7.69(m,2H),7.31(d,J=7.2Hz,1H),7.20(s,2H),6.48(s,1H),3.56-3.44(m, 4H),3.31(s,4H),3.06(d,J=6.5Hz,2H),2.89(s,2H),2.75(d,J=5.8Hz,4H),2.68-2. 62(m,2H),2.19(s,2H),2.05(s,2H),1.88(dt,J=12.0,5.4Hz,6H),1.78-1.63(m,2H). 13C-NMR (100MHz, CDCl3) δ161.31,157.51,151.65,149.60,144.45,138.01,134.47,131.98,130.79,130.68,128.81,128.61,127.87,125.28, 123.68,121.75,121.17,120.26,119.84,114.96,103.51,49.73,49.3 2,49.12,26.74,26.60,24.86,20.35,20.31,19.41,18.84,18.60.HRMS m / z:calcd for C 39 H 39 N2O3 + [M] + :m / z583.2955; found:m / z 583.2963.

[0036]

[0037] Example 2

[0038] Responsiveness of the probe P2 prepared in Example 1 of the present invention to viscosity

[0039] Probe P2 was prepared into a 1 mM stock solution using dimethyl sulfoxide. Glycerol and pH 7.4 phosphate buffer were mixed in different proportions to adjust the viscosity of the sample. The probe was dissolved in solutions of different viscosities and the final concentration of the probe was maintained at 10 μM. Figure 1 As shown in Figure A, as the volume ratio of glycerol in the medium increases (glycerol increases from 0% to 99%), the fluorescence intensity of the probe at 819 nm gradually increases. In addition, the logarithmic value of the fluorescence intensity of the probe at 819 nm shows a good linear relationship with the logarithmic value of the viscosity, as shown in Figure 2. Figure 1 As shown in B.

[0040] Example 3

[0041] Responsiveness of probe P2 prepared in Example 1 of the present invention to peroxynitrite

[0042] In a phosphate buffer containing probe P2 at a concentration of 10 μM, as the concentration of peroxynitrite increased (from 0 to 30 μM), the fluorescence intensity of the probe at 511 nm gradually increased. Figure 2 In addition, there is a good linear relationship between the fluorescence intensity of the probe at 511 nm and the concentration of peroxynitrite, ONOO - The detection limit is 18nM. Figure 2 As shown in B.

[0043] Example 4

[0044] Selectivity of probe P2 prepared in Example 1 of the present invention

[0045] In the phosphate buffer solution of probe P2 at a concentration of 10 μM, common ions, reactive oxygen species, and amino acids were added to investigate the selectivity of the probe for viscosity and peroxynitrite. Figure 3 As shown in A, after adding the above substances, the fluorescence signal of the probe at 819nm hardly changed, but it showed bright fluorescence in glycerol, indicating that the probe has high selectivity for viscosity. Similarly, these species did not cause the fluorescence of the probe at 511nm to change, while the presence of peroxynitrite can significantly increase the fluorescence, as shown in Figure 3 As shown in B, it shows that the probe can specifically recognize peroxynitrite.

[0046] Example 5

[0047] Probe P2 prepared in Example 1 of the present invention is used for cell viscosity imaging

[0048] SK-OV-3 cells were divided into three groups: the first group was treated with 10 μM probe P2 for 30 min (control group); the second group was treated with 25 μM nystatin for 30 min, and then treated with 10 μM probe for 30 min; the third group was treated with serum-free incomplete culture medium for 30 min, and then treated with 10 μM probe for 30 min. The cells were then irradiated with 640 nm excitation light, and the fluorescence signals at 650-750 nm were collected. Figure 4 As shown in A, compared with the control group, the cells in the second and third groups showed stronger fluorescence signals. Figure 4 B is the statistical value of the fluorescence intensity of the cells. The experimental results show that the probe can be used for imaging analysis of cell viscosity.

[0049] Example 6

[0050] Probe P2 for cellular peroxynitrite imaging

[0051] HeLa cells were divided into four groups: the first group used 5 μM probe P2 to incubate the cells for 30 minutes (control group); the second group used 200 μM lindane to treat the cells for 4 hours, and then incubated the cells with 5 μM probe for 30 minutes; the third group used 1 μg / mL lipopolysaccharide and 100 ng / mL interferon γ to culture the cells for 8 hours, and then incubated the cells with 5 μM probe for 30 minutes; the fourth group used 1 μg / mL lipopolysaccharide and 100 ng / mL interferon γ to culture the cells for 8 hours, and then cultured the cells with 10 nM phorbol ester for 30 minutes, and then incubated the cells with 5 μM probe for 30 minutes. Finally, the cells were irradiated with 405 nm excitation light, and the fluorescence signals of 500-540 nm were collected. Figure 5 As shown in A, compared with the control group, the cells in the second, third and fourth groups showed stronger fluorescence. Figure 5 B is the statistical value of the fluorescence intensity of the cell. The experimental results show that the probe can be used for imaging analysis of intracellular peroxynitrite.

[0052] Example 7

[0053] Probe P2 for imaging viscosity and peroxynitrite during ferroptosis

[0054] SK-OV-3 cells were divided into two groups: the first group was treated with 5 μM probe P2 for 30 min (control group); the second group was treated with 20 μM elastin for 12 h, and then incubated with 5 μM probe for 30 min. The cells were then irradiated with 405 nm and 640 nm excitation light, and the fluorescence signals at 500-540 nm and 650-750 nm were collected in turn. Figure 6 As shown in A, cells undergoing ferroptosis showed stronger red and green fluorescence compared with the control group, indicating that the ferroptosis process is accompanied by higher viscosity and higher levels of peroxynitrite. Figure 6 B is the statistical value of the fluorescence intensity of the cells. The experimental results show that the probe can simultaneously monitor the changes in viscosity and peroxynitrite levels during cell ferroptosis.

[0055] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A dual-response fluorescent probe, characterized in that: The chemical structure is as follows:

2. The method for preparing a dual-response fluorescent probe according to claim 1, wherein: The following steps are involved: (1) Dissolving 8-hydroxyjulolidine and phthalic anhydride in toluene, heating under reflux, removing the solvent, and performing chromatography to obtain compound 3; (2) After the reaction of compound 3 and cyclohexanone in concentrated sulfuric acid, the mixture was poured into crushed ice and stirred, and perchloric acid was added and stirred. After standing, the mixture was filtered to obtain compound 4; (3) Compound 4 and 9-formyljulolidine were dissolved in acetic anhydride, heated to reflux, the solvent was removed, and the fluorescent probe P2 was obtained by chromatography; 3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of 8-hydroxyjulolidine to phthalic anhydride is 1:1-2; the reaction temperature is controlled to be 80-100° C., and the reaction is carried out for 8-24 hours; the chromatography is performed using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-50 as the eluent.

4. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of cyclohexanone, compound 3 and concentrated sulfuric acid is 1:1-2:30-100; the reaction temperature of compound 3 and cyclohexanone is 60-90°C, and the reaction time is 1-3h; the mass ratio of concentrated sulfuric acid to crushed ice is 1:10-30, the temperature is controlled at 0°C, and the stirring time is 2-10min; the mass fraction of perchloric acid is 50%-70%, the molar ratio of perchloric acid to compound 3 is 1:5-10, and the stirring time is 10-20min; and the standing time is 1-3h.

5. The preparation method according to claim 2, characterized in that: In step (3), the molar ratio of compound 4 to 9-aldehyde julolidine is 1:1-2; the reaction temperature is 80°C-100°C, and the reaction time is 2-8h; the chromatography is performed by using a silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20-80 as the eluent.

6. The dual-response fluorescent probe as claimed in claim 1 is used for qualitative or quantitative detection of ONOO - It is used as a detection reagent for ions and / or viscosity.

7. Use of the dual-response fluorescent probe as claimed in claim 1 in preparing a peroxynitrite and / or viscosity fluorescent imaging agent in living cells.

8. Use of the dual-response fluorescent probe as claimed in claim 1 in preparing a fluorescent imaging agent in the process of cell ferroptosis.

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