CN113956274A - Design and synthesis method of fluorescent probe capable of responding to viscosity and peroxynitrite change in epileptic diseases - Google Patents
Design and synthesis method of fluorescent probe capable of responding to viscosity and peroxynitrite change in epileptic diseases Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 50
- 230000008859 change Effects 0.000 title claims abstract description 14
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 title claims abstract 5
- 201000010099 disease Diseases 0.000 title abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title abstract description 6
- 230000001037 epileptic effect Effects 0.000 title abstract description 5
- 238000013461 design Methods 0.000 title description 2
- 238000001308 synthesis method Methods 0.000 title description 2
- 230000004044 response Effects 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000009977 dual effect Effects 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims description 10
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 229940125782 compound 2 Drugs 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
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- BATKIZWNRQGSKE-UHFFFAOYSA-N 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethanone Chemical compound C1=CC(C(=O)C)=CC=C1B1OC(C)(C)C(C)(C)O1 BATKIZWNRQGSKE-UHFFFAOYSA-N 0.000 claims description 2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1007—Non-condensed systems
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
Abstract
The invention relates to the technical field of fluorescent probes, in particular to a dual-response fluorescent probe for simultaneously detecting peroxynitrite and viscosity change in an epileptic disease process and a preparation method thereof. The synthesized double-response fluorescent probe compound for detecting peroxynitrite and viscosity change has the following structure, and is simple to synthesize and easy to obtain in reaction.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to a dual-response fluorescent probe for simultaneously detecting peroxynitrite and viscosity change in an epileptic disease process and a preparation method thereof.
Background
Epilepsy is a chronic neurodegenerative disease characterized by recurrent, unpredictable tics. During the process of occurrence and development of epilepsy, a great deal of activity is continuously generatedOxygen (ROS), which further reacts with Nitric Oxide (NO) to form RNS, such as peroxynitrite (ONOO)-) And thus may further lead to neuronal cell death by binding to a number of biologically active molecules, including proteins, nucleic acids and lipids. Overexpressed ONOO-Is considered as a key neurotoxicity factor, plays an important role in the pathogenesis of epilepsy and can be used as a potential biomarker for early prediction of epilepsy. However, ONOO-The potential biological role in epileptogenesis is not fully understood. Therefore, to explore the in vivo ONOO-Pathophysiological mechanisms of-And study its role in epilepsy, develop effective imaging tools to monitor ONOO in the brain-It is of great importance.
Cell viscosity is an important analyte at the cellular level, and changes in viscosity can reflect interactions between biomolecules and the transport of metabolic waste products. Studies have shown that intracellular perturbations can be induced by altering the motility of intracellular biomolecules. These perturbations can affect signal transduction and transport of intracellular species, as well as diffusion of short-lived reactive intermediates, such as the diffusion of ROS during oxidative stress. ROS also alter cell viscosity by oxidizing species. Abnormal changes in intracellular viscosity may lead to diseases such as malignancy, alzheimer's disease, parkinson's disease, etc., and therefore it is of paramount importance to develop effective imaging tools for monitoring the detection of cell viscosity.
Fluorescence imaging has been widely used in the fields of biomedical and clinical diagnostics. Near-infrared (NIR, 700 and 1700nm) fluorescence imaging develops biological tissues in the NIR band, and is more favorable for improving the signal-to-noise ratio and the sensitivity of imaging compared with the traditional fluorescence imaging in the visible light band (400 and 760 nm). High quality fluorescence imaging requires the use of good fluorescent probes, and the rapid development of nanotechnology has led to the emergence of organic dyes with good fluorescence properties. Compared with inorganic fluorescent probes, the organic fluorescent probe has the advantages of high safety, good biocompatibility, strong optical stability and the like. Therefore, NIR fluorescence imaging assisted by organic fluorescent probes can provide structural and dynamic information of biological samples for researchers, and is the current lightThe hot spot of the multidisciplinary cross-research fields of science, chemistry, biomedicine and the like. Accurate tracking of tumor-specific behavior in vivo with a probe is a highly desirable strategy. However, few probes are available for simultaneous detection of intracellular viscosity and ONOO-. Currently, simultaneous detection of viscosity and ONOO is mostly achieved by using two probes-. However, this method has disadvantages such as photobleaching, uneven probe loading, and low efficiency. Therefore, the development of a dual-response fluorescent probe for simultaneously detecting peroxynitrite and viscosity change in the epileptic disease process is urgently needed.
Disclosure of Invention
The invention aims to solve the problem that the existing traditional method for detecting ONOO-The instrument limit, the cost is too high and the like; when the fluorescence probe method is used for detection, the simultaneous detection of ONOO by a single probe cannot be realized-And viscosity. The invention provides a fluorescent probe capable of responding to peroxynitrite and viscosity change based on acetylphenylboronic acid pinacol ester and a preparation method thereof.
The structural formula of the fluorescent probe is as follows:
the synthetic route is as follows:
the preparation method of the fluorescent probe comprises the following steps:
And 3, dissolving the compound 2 and the (3, 5, 5-trimethylcyclohex-2-enylidene) malononitrile into absolute ethyl alcohol, dropwise adding a catalytic amount of piperidine, and stirring for 8 hours at 80 ℃. The solvent was removed under reduced pressure and the product was purified by silica gel column to give compound 3.
Drawings
FIG. 1 fluorescence spectra of fluorescent probes in different viscosity systems
FIG. 2 fluorescent probes and different concentrations of ONOO-Fluorescence spectrum after reaction
FIG. 3 fluorescence spectra of fluorescent probes in different solvent systems
FIG. 4 fluorescent response of fluorescent probes (10. mu.M) to different substrates (50. mu.M each) includes: (1) OH (2)1O2 (3)H2O2 (4) TBHP (5)TBO (6)NO，(7)NO2 - (8)NO3 - (9)Cys，(10)GSH (11)Hcy (12)Br-(13)Cl-(14)CO3 2- (15)F- (16)HCO3 - (17)HSO3 - (18)I- (19)SO3 2- (20)Fe2+ (21)Mg2+ (22)Zn2+(23)ONOO-
FIG. 5 fluorescent probes (10. mu.M) comprising, on different substrates (50. mu.M each): (1) OH (2)1O2 (3)H2O2 (4)TBHP (5) TBO (6)NO，(7)NO2 - (8)NO3 - (9)Cys，(10)GSH (11)Hcy (12)Br- (13)Cl- (14)CO3 2- (15)F- (16)HCO3 - (17)HSO3 - (18)I- (19)SO3 2- (20)Fe2+ (21)Mg2+ (22)Zn2+ (23)ONOO-In the presence of (C) to ONOO-Fluorescence response of
FIG. 6 fluorescent probes against ONOO in tumor cells-Fluorescence imaging of-. (a) Control group (b) 10. mu.M ONOO-group (c)1mg/mL LPS and 50ng/M IFN-. gamma.group
FIG. 7 nuclear magnetic hydrogen spectroscopy data for the probe
Nuclear magnetic carbon spectral data of the probe of FIG. 8
The first embodiment is as follows: a fluorescence spectrum experiment for detecting viscosity change by a fluorescent probe comprises the following steps:
step one, preparation of stock solution
1mg of the fluorescent probe compound was weighed and dissolved in DMSO to prepare a DMSO solution of 1mM of the fluorescent probe.
Methanol and glycerol were formulated as shown in the table below to give stock solutions of different viscosities.
Step two, testing spectral performance
Adding 2 mu L of DMSO solution of fluorescent probe into stock solutions with different viscosities and the same volume respectively to form a 200 mu L mixed system, and recording the fluorescence spectrum change before and after reaction after shaking for 30 min.
FIG. 1 shows fluorescence spectra of fluorescent probes in different viscosity systems. As shown in FIG. 1, the fluorescence intensity greatly increases with increasing viscosity, i.e., the fluorescent probe responds to changes in viscosity.
The second embodiment is as follows: the fluorescence spectrum experiment for detecting ONOO-by the fluorescent probe comprises the following steps:
step one, preparation of stock solution
1mg of the fluorescent probe compound was weighed and dissolved in DMSO to prepare a DMSO solution of 1mM of the fluorescent probe.
Adding 5ml, 0.6M sodium nitrite solution into 5ml, 0.6M hydrogen peroxide solution, stirring at high speed with magnetic stirrer, rapidly adding 0.6g sodium hydroxide, reacting for 2min, adding 0.1g manganese dioxide, removing unreacted hydrogen peroxide, freezing for storage, and passing 0.1M sodium hydroxide solution as reference to obtain ONOO with concentration of 0.2mM-And (4) mother liquor.
Step two, testing spectral performance
mu.L of DMSO solutions of fluorescent probes were added to 100. mu.L of PBS buffer, and different volumes of ON were addedOO-And (5) adding deionized water into the mother liquor, supplementing the volume of the mixed system to 200 mu L, vibrating the Yao for 30min, and recording the change of fluorescence spectra before and after reaction.
FIG. 2 shows fluorescent probes and different concentrations of ONOO-Fluorescence spectrum after reaction. As can be seen from FIG. 2, following the ONOO-The concentration is increased and the fluorescence intensity is gradually increased, i.e. the fluorescence probe is directed to ONOO-There is a response.
The third concrete implementation mode: fluorescence spectrum experiments of fluorescent probes in different solvents comprise the following steps:
step one, preparation of stock solution
1mg of the fluorescent probe compound was weighed and dissolved in DMSO to prepare a DMSO solution of 1mM of the fluorescent probe.
Step two, testing spectral performance
mu.L of a DMSO solution of the fluorescent probe was added to the same volume of different solvents.
FIG. 3 shows fluorescence spectra of fluorescent probes in different solvents.
The fourth concrete implementation mode: fluorescent probe pair ONOO-Selectivity and interference rejection
Step one, preparation of stock solution
1mg of the fluorescent probe compound was weighed and dissolved in DMSO to prepare a DMSO solution of 1mM of the fluorescent probe.
Adding 5ml, 0.6M sodium nitrite solution into 5ml, 0.6M hydrogen peroxide solution, stirring at high speed with magnetic stirrer, rapidly adding 0.6g sodium hydroxide, reacting for 2min, adding 0.1g manganese dioxide, removing unreacted hydrogen peroxide, freezing for storage, and passing 0.1M sodium hydroxide solution as reference to obtain ONOO with concentration of 0.2mM-And (4) mother liquor.
The ROS/RNS (. OH; R) of the same concentration are prepared,1O2、H2O2、TBHP、TBO、NO、NO2 -、NO3 -) Biological thiols (Cys, GSH, Hcy), common anions (Br)-，Cl-，CO3 2-，F-，HCO3 -，HSO3 -，I-，SO3 2-) Metal ion (Fe)2+， Mg2+，Zn2+) And (3) solution.
Step two, testing spectral performance
1. Fluorescent probe pair ONOO-Selectivity of (2)
Adding 2 mu L of DMSO solution of a fluorescent probe into 100 mu L of PBS buffer solution, adding 50 mu L of solutions with the same concentration and containing different substrates, adding deionized water to supplement the volume of the mixed system to 200 mu L, vibrating for 30min, and recording the change of fluorescence spectrum before and after reaction.
FIG. 4 is a graph showing the fluorescence response of a fluorescent probe (10. mu.M) to different substrates (50. mu.M) including: (1) OH (2)1O2 (3)H2O2 (4)TBHP (5)TBO (6)NO (7)NO2 - (8)NO3 - (9)Cys (10)GSH (11)Hcy (12)Br- (13)Cl-(14)CO3 2- (15)F- (16)HCO3 - (17)HSO3 - (18)I- (19)SO3 2- (20)Fe2+ (21)Mg2+ (22)Zn2+(23)ONOO-
As shown in FIG. 4, only ONOO-The induced fluorescence is significantly enhanced.
2. Anti-interference effect of fluorescent probe
mu.L of DMSO solutions of fluorescent probes were added to 100. mu.L of PBS buffer, and 10. mu.L of solutions containing different substrates (. OH, g,1O2、H2O2、TBHP、TBO、NO、NO2 -、NO3 -、Cys、GSH、Hcy、Br-，Cl-，CO3 2-， F-，HCO3 -，HSO3 -，I-，SO3 2-、Fe2+，Mg2+，Zn2+) Adding 78 μ L deionized water, shaking for 30min, and adding 10 μ L ONOO-And (5) vibrating the mother liquor again for 30min, and recording the fluorescence change before and after the reaction.
As can be seen from FIG. 5, when ONOO is used-(10. mu.M) together with other substratesUpon addition, the fluorescence intensity still increased significantly.
In conclusion, the fluorescent probe is highly selective for ONOO-.
The fifth concrete implementation mode: fluorescent probes corresponding to applications in ONOO-In situ detection of overexpressed tumor cells
As shown in FIG. 6, (a) when the cells were incubated with the fluorescent probe (10. mu.M) for 15min, there was almost no fluorescence. However, (b) when the cells were incubated with the probe for 15 minutes, then the probes were incubated with ONOO-After 30min of treatment (10. mu.M), the cells showed an increase in fluorescence. (c) Using Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), stimulating cells to produce endogenous ONOO-. A large fluorescence increase occurred when tumor cells were incubated with LPS and IFN-. gamma.for 4h and then with the probe for 15 min. All results clearly indicate that the probes can be used for both exogenous and endogenous ONOO in living cells-And (6) imaging.
Although the preferred embodiments of the present invention and the evaluation of biological activity have been described in detail, the present invention is not limited to the details of the above embodiments, and various equivalent changes can be made to the technical solution of the present invention within the technical spirit of the present invention, and these equivalent changes are within the scope of the present invention. It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (2)
1. A peroxynitrite and viscosity change dual-response fluorescent probe is characterized by having a structure shown in the general formula I:
2. the method for preparing a fluorescent probe for detecting peroxynitrite and viscosity change as set forth in claim 1, which comprises the steps of:
step 1, dissolving 4-acetylphenylboronic acid pinacol ester and 5-bromo-2-furfural in absolute ethyl alcohol, dropwise adding a catalytic amount of piperidine, and stirring at 80 ℃ for 8 hours. Removing the solvent under reduced pressure, purifying the product with silica gel column to obtain compound 2
Step 2, dissolving the compound 1 in dimethyl ether, and adding 4-formyl phenylboronic acid and Na2CO3\Pd(PPh3)4Stirring at 80 ℃ for 12h under a nitrogen atmosphere. Cooling, filtering, adding ethyl acetate into the filtrate, washing with saturated brine, removing the solvent under reduced pressure, and purifying the product with silica gel column to obtain compound 2
And 3, dissolving the compound 2 and the (3, 5, 5-trimethylcyclohex-2-enylidene) malononitrile into absolute ethyl alcohol, dropwise adding a catalytic amount of piperidine, and stirring for 8 hours at 80 ℃. The solvent was removed under reduced pressure and the product was purified by silica gel column to give compound 3.
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| CN113045596A (en) * | 2021-03-24 | 2021-06-29 | 台州学院 | Peroxynitroso anion and viscosity dual-response type fluorescent probe and preparation and application thereof |
| CN113480458A (en) * | 2021-07-05 | 2021-10-08 | 西北大学 | Hydrogen sulfide donor, preparation method and application thereof |
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| CN113045596A (en) * | 2021-03-24 | 2021-06-29 | 台州学院 | Peroxynitroso anion and viscosity dual-response type fluorescent probe and preparation and application thereof |
| CN113480458A (en) * | 2021-07-05 | 2021-10-08 | 西北大学 | Hydrogen sulfide donor, preparation method and application thereof |
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| BINGPENG GUO等: ""A lysosome targetable versatile fluorescent probe for imaging viscosity and peroxynitrite with different fluorescence signals in living cells"", 《J.MATER.CHEM.B》, vol. 6, pages 580 - 585 * |
| PAN JIA等: ""Dicyanoisophorone-Derived Near-Infrared Fluorescent Probe for Ultrasensitive Detection of Peroxynitrite in Living Cells and Zebrafish"", 《IND.ENG.CHEM.RES.》, vol. 58, pages 19778 - 19784 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115772096A (en) * | 2022-12-02 | 2023-03-10 | 安徽大学 | Two-photon fluorescent probe for two-channel detection and preparation method and application thereof |
| CN115772096B (en) * | 2022-12-02 | 2024-03-01 | 安徽大学 | Two-photon fluorescent probe for double-channel detection and preparation method and application thereof |
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