CN109608414B - Fluorescent probe for detecting peroxynitrite and preparation method and application thereof - Google Patents
Fluorescent probe for detecting peroxynitrite and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 16
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- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
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Abstract
The invention belongs to the technical field of synthesis and detection, and particularly relates to a fluorescent probe for detecting peroxynitrite as well as a preparation method and application thereof. The invention provides a universal recognition group for detecting peroxynitrite, and three new fluorescent probes are constructed by connecting the universal recognition group with a common fluorophore, are respectively named as BT-FAP, NA-FAP and NR-FAP, and can realize the detection with ONOO by respectively taking benzothiazole-aminonaphthalene (BT), Naphthalimide (NA) and benzophenoxazine (NR) as fluorophores and Formamide (FAP) as recognition groups‑Transient response (both<4s), high sensitivity and good selectivity, and is very suitable for ONOO in cells‑Fluorescence imaging studies of (1).
Description
Technical Field
The invention belongs to the technical field of synthesis and detection, and particularly relates to a formamide recognition group-based fluorescent probe for detecting peroxynitrite, and a preparation method and application thereof.
Background
In vivo, peroxynitrite (ONOO)-) Is an important active nitrogen species, is generated by the reaction of superoxide radical anions and nitric oxide, and can simultaneously represent the level of oxidative stress and nitrification stress. Moreover, existing studies have shown that ONOO-Is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, diabetes, cancer, and the like. ONOO-Short half-life and extremely low steady-state concentrations under physiological conditions, allowing detection of ONOO in living cells and tissues-It becomes very difficult. Fluorescence analysis based on fluorescent probes enables visualization of molecular events in real time in cells and animal models, providing an effective assay for the above problems. However, the detection of ONOO has been reported at present-The following problems are common to the fluorescent probes of (1): (1) the identification group has no universal applicability, is difficult to popularize to other fluorescent groups, and increases the design difficulty of a new probe; (2) the probe has complex chemical structure, long synthesis route and difficult synthesis, and can not realize large-scale production. These problems have led to the existing ONOO-The practical application of the fluorescent probe is greatly limited, and the large-scale popularization and application are not facilitated, so that the development of a new fluorescent probe is necessaryFor detecting ONOO-The fluorescent probe and the preparation method thereof.
Disclosure of Invention
In view of the problems in the prior art, the present invention aims to provide a fluorescent probe for detecting peroxynitrite, a preparation method and applications thereof. The invention provides a universal recognition group for detecting peroxynitrite, and three new fluorescent probes are constructed by connecting the universal recognition group with a common fluorophore, are respectively named as BT-FAP, NA-FAP and NR-FAP, and can realize the detection with ONOO by respectively taking benzothiazole-aminonaphthalene (BT), Naphthalimide (NA) and benzophenoxazine (NR) as fluorophores and Formamide (FAP) as recognition groups-Transient response (both<4s), high sensitivity and good selectivity, and is very suitable for ONOO in cells-Fluorescence imaging studies of (1).
One of the purposes of the invention is to provide three fluorescent probes for detecting peroxynitrite based on formamide recognition groups.
The invention also aims to provide a preparation method of the fluorescent probe for detecting peroxynitrite based on the formamide recognition group.
The invention also aims to provide an imaging method of the fluorescent probe for detecting peroxynitrite based on the formamide recognition group in the liver cancer cells.
The fourth purpose of the invention is to provide a formamide recognition group-based fluorescent probe for detecting peroxynitrite and application of the fluorescent probe in an imaging method of liver cancer cells.
In order to achieve the above purpose, the invention specifically discloses the following technical scheme:
the invention discloses a first formamide recognition group-based fluorescent probe for detecting peroxynitrite, which is named as BT-FAP and has a structural formula as follows:
the invention discloses a second formamide-recognition-group-based fluorescent probe for detecting peroxynitrite, which is named as NA-FAP and has the structural formula as follows:
thirdly, the invention discloses a third formamide recognition group-based fluorescent probe for detecting peroxynitrite, which is named as NR-FAP and has the structural formula:
secondly, the invention discloses a preparation method of the three fluorescent probes for detecting peroxynitrite based on the formamide recognition group, wherein the reaction equations are respectively as follows:
wherein the compound 1 represents benzothiazole-aminonaphthalene, the compound 2 represents naphthalimide, and the compound 3 represents benzophenoxazine.
The reaction steps are as follows: respectively dissolving the compound 1, 2 or 3 in the reaction equation in formic acid, uniformly stirring to obtain a mixed solution, and reacting for a set time to obtain the compound.
Further, in the above reaction step, the ratio of the compound 1, 2 or 3 to formic acid is: 0.2-1.5 mM: 0.5-5 mL.
Further, in the reaction step, the reaction temperature is 60-100 ℃, and the reaction time is 3-6 h; preferably, the reaction temperature is 75-85 ℃ and the reaction time is 4.5-5 h.
Further, the reaction step further comprises the steps of spin-drying and purifying the reaction product solution. Preferably, the reaction product is purified by silica gel column chromatography.
More preferably, the eluent for silica gel column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 80-130:1, 75-125:1, 40-60: 1; preferably 100:1, 100:1, 50:1, respectively.
In addition, the invention also discloses the ONOO in living cells by the formamide recognition group-based fluorescent probe for detecting peroxynitrite-Application in imaging. Specifically, the application is an imaging method of a fluorescent probe in liver cancer cells, and the imaging method comprises the following steps: culturing hepatocarcinoma cell with ONOO-And (3) after incubation of the release agent, incubating with a dimethyl sulfoxide solution of the probe, after incubation for a set time, removing the dimethyl sulfoxide solution of the probe, washing with PBS, and then performing laser confocal imaging to obtain the probe.
Preferably, in the above imaging method, the ONOO-The release agent is SIN-1.
Preferably, in the above imaging method, the incubation time is 20-40 min.
Finally, the invention discloses the formamide recognition group-based fluorescent probe for detecting peroxynitrite and application of the imaging method of the formamide recognition group-based fluorescent probe in liver cancer cells in the field of biological detection.
Compared with the prior art, the invention has the beneficial effects that:
(1) the three probe molecules provided by the invention have the maximum fluorescence emission wavelengths respectively positioned at 500nm, 560nm and 620nm, have moderate Stokes shift (about 50nm), can effectively reduce self-absorption and improve the imaging accuracy.
(2) The three probe molecules provided by the invention can realize the alignment of ONOO-Quick response (all reaction completed within 4s) and to ONOO-Has high sensitivity and selectivity, good staining effect of the probe in living cells, short staining time (20min), high staining efficiency, and suitability for ONOO in cells-Fluorescence imaging studies of (1).
(3) The three probe molecules provided by the invention can realize the ONOO in the liver cancer cells incubated by SIN-1-An increase in concentration, thereby causingTo detect ONOO in living cells and tissues-The real-time visualization of molecular events in cells and animal models by fluorescence analysis is realized more easily.
(4) The synthesis steps of the three probe molecules are simple, the yield is high, and the purification is easy; in addition, in the preparation method, formic acid is directly used as a reactant, and meanwhile, formic acid is used as a solvent, so that other auxiliary solvents or reagents are not used, the cost is low, and the environmental pollution is small.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 shows the three fluorescent probes of example 1 for different concentrations of ONOO-A fluorescence response spectrum of (a); the abscissa is the wavelength (nm) and the ordinate is the fluorescence emission intensity.
FIG. 2 shows the fluorescence intensity at 500nm, 560nm and 620nm of the three fluorescent probes of example 1 with different concentrations of ONOO-The linear relationship of (a); the abscissa is ONOO-Concentration (. mu.M), the ordinate is the fluorescence emission intensity of the probe at the corresponding wavelengths (500nm, 560nm and 620 nm).
FIG. 3 is a graph of the change in fluorescence intensity at 500nm, 560nm and 620nm, respectively, with time for the three fluorescent probes of example 1 in real time; the abscissa is time(s) and the ordinate is the fluorescence emission intensity of the three probes at the corresponding wavelengths (500nm, 560nm and 620 nm).
FIG. 4 is a graph showing fluorescence intensities at 500nm, 560nm and 620nm of three kinds of fluorescent probes of example 1 in the presence of different kinds of substances, respectively. The abscissa is the different species and the ordinate is the fluorescence emission intensity of the probe at the corresponding wavelengths (500nm, 560nm and 620 nm).
FIG. 5 is a graph of fluorescence images of the three fluorescent probes of example 1 in hepatoma cells (HepG 2).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced by the background, currently, there is a detection ONOO-The following problems are common to the fluorescent probes of (1): (1) the identification group has no universal applicability, is difficult to popularize to other fluorescent groups, and increases the design difficulty of a new probe; (2) the probe has complex chemical structure, long synthesis route and difficult synthesis, and can not realize large-scale production. Therefore, the invention provides a fluorescent probe for detecting peroxynitrite and a preparation method and application thereof, and the invention is further explained by combining the attached drawings and the specific embodiment.
It should be noted that: in the following examples, the present invention utilizes the existing raw material compounds 1, 2, 3, and prepares three probes BT-FAP, NA-FAP and NR-FAP through simple and efficient synthesis in one step and separation by silica gel chromatographic column. For example, compound 1 is obtained from 6-amino-2-naphthoic acid and o-aminothiophenol in polyphosphoric acid, heated to 150 ℃, and reacted overnight. Compound 2 is described in Cui, l.; peng, z.; ji, c.; huang, j.; huang, d.; ma, j.; zhang, s.; qian, x.; xu, Y.Chem.Commun.2014,50, 1485-1487. Compound 3 is described in Greenspan, p.; fowler, S.D.J.lipid Res.1985,26, 781-789.
Example 1
Three preparation methods of a fluorescent probe for detecting peroxynitrite based on a formamide recognition group have the following reaction equations:
wherein, the compound 1 represents benzothiazole-aminonaphthalene, the compound 2 represents naphthalimide, and the compound 3 represents benzophenoxazine.
Specifically, the preparation steps of the three fluorescent probes in this embodiment are as follows:
(1) 1mmol of compound 1 or 2 or 3 (274 mg or 268mg or 263mg, respectively) was mixed with 3mL of formic acid, respectively;
(2) then, heating the three mixed solutions obtained in the step (1) to 80 ℃ and reacting for 5 hours;
(3) after the reaction is finished, cooling the three reaction liquids to room temperature, then concentrating and spin-drying the solvent, and performing column chromatography separation on the obtained solids to respectively obtain three product solids: BT-FAP, NA-FAP and NR-FAP, namely the fluorescent probe for detecting peroxynitrite based on the formamide recognition group; wherein, the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 100:1, 100:1 and 50: 1.
Through detection, the quality of BT-FAP, NA-FAP and NR-FAP obtained in the embodiment is 243mg, 257mg and 235mg respectively; corresponding to yields of 80%, 87%, 81%, respectively.
Example 2
The reaction equation/principle of three methods for preparing the fluorescent probe for detecting peroxynitrite based on the formamide recognition group is the same as that in example 1, and the specific preparation steps are as follows:
(1) mixing 1.2mmol of compound 1 or 2 or 3 with 5mL of formic acid respectively;
(2) then, heating the three mixed solutions obtained in the step (1) to 60 ℃ and reacting for 6 hours;
(3) after the reaction is finished, cooling the three reaction liquids to room temperature, then concentrating and spin-drying the solvent, and performing column chromatography separation on the obtained solids to respectively obtain three product solids: BT-FAP, NA-FAP and NR-FAP, namely the fluorescent probe for detecting peroxynitrite based on the formamide recognition group; wherein, the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 80:1, 110:1 and 40: 1.
Through detection, the yields of BT-FAP, NA-FAP and NR-FAP obtained in the embodiment are 84%, 86% and 82% respectively.
Example 3
The reaction equation/principle of three methods for preparing the fluorescent probe for detecting peroxynitrite based on the formamide recognition group is the same as that in example 1, and the specific preparation steps are as follows:
(1) mixing 1.5mmol of compound 1 or 2 or 3 with 4mL of formic acid respectively;
(2) then, heating the three mixed liquids obtained in the step (1) to 85 ℃ and reacting for 4.5 hours;
(3) after the reaction is finished, cooling the three reaction liquids to room temperature, then concentrating and spin-drying the solvent, and performing column chromatography separation on the obtained solids to respectively obtain three product solids: BT-FAP, NA-FAP and NR-FAP, namely the fluorescent probe for detecting peroxynitrite based on the formamide recognition group; wherein, the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 120:1, 125:1 and 55: 1.
Through detection, the yields of BT-FAP, NA-FAP and NR-FAP obtained in the embodiment are 87%, 84% and 85% respectively.
Example 4
The reaction equation/principle of three methods for preparing the fluorescent probe for detecting peroxynitrite based on the formamide recognition group is the same as that in example 1, and the specific preparation steps are as follows:
(1) mixing 0.2mmol of compound 1 or 2 or 3 with 0.5mL of formic acid, respectively;
(2) then, heating the three mixed solutions obtained in the step (1) to 100 ℃ and reacting for 3 hours;
(3) after the reaction is finished, cooling the three reaction liquids to room temperature, then concentrating and spin-drying the solvent, and performing column chromatography separation on the obtained solids to respectively obtain three product solids: BT-FAP, NA-FAP and NR-FAP, namely the fluorescent probe for detecting peroxynitrite based on the formamide recognition group; wherein, the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 130:1, 90:1 and 60:1 in sequence.
Through detection, the yields of BT-FAP, NA-FAP and NR-FAP obtained in the embodiment are 82%, 88% and 83% respectively.
Example 5
The reaction equation/principle of three methods for preparing the fluorescent probe for detecting peroxynitrite based on the formamide recognition group is the same as that in example 1, and the specific preparation steps are as follows:
(1) mixing 0.5mmol of compound 1 or 2 or 3 with 1mL of formic acid respectively;
(2) then, heating the three mixed solutions obtained in the step (1) to 75 ℃ and reacting for 4.5 hours;
(3) after the reaction is finished, cooling the three reaction liquids to room temperature, then concentrating and spin-drying the solvent, and performing column chromatography separation on the obtained solids to respectively obtain three product solids: BT-FAP, NA-FAP and NR-FAP, namely the fluorescent probe for detecting peroxynitrite based on the formamide recognition group; wherein, the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 90:1, 75:1 and 45: 1.
Through detection, the yields of BT-FAP, NA-FAP and NR-FAP obtained in the embodiment are 86%, 83% and 87% respectively.
Product performance testing, three fluorescent probes BT-FAP, NA-FAP and NR-FAP prepared in example 1 were tested, and the results are shown below:
(1) mass spectrum and nuclear magnetism characterization:
BT-FAP: a pair of rotamers.1H NMR(400MHz,DMSO-d6):10.56(s,0.7H),10.49(d,J＝10.9Hz,0.3H),9.05(d,J＝10.8Hz,0.3H),8.63(s,1H),8.41(d,J＝9.2Hz,1.7H),8.20-8.12(m,2.7H),8.09(d,J＝8.1Hz,1H),8.02(d,J＝8.6Hz,0.7H),7.97(d,J＝8.5Hz,0.3H),7.78(s,0.3H),7.68(d,J＝8.7Hz,0.7H),7.60-7.52(m,1.3H),7.48(t,J＝7.4Hz,1H).13CNMR(100MHz,DMSO-d6):167.35,162.73,160.11,153.66,137.88,137.49,135.02,134.85,134.48,130.53,129.86,129.55,129.10,128.93,128.47,128.06,127.25,127.08,126.68,125.49,124.73,124.52,122.78,122.35,120.68,119.38,115.19,112.36.HRMS(ESI):calculated for C18H13N2OS+(M+H+)305.0743,found 305.0746.
NA-FAP:1H NMR(400MHz,DMSO-d6):10.96(s,1H),8.66(d,J＝6.7Hz,2H),8.45(d,J＝25.5Hz,3H),7.87(s,1H),4.01(br s,2H),1.70-1.50(m,2H),1.41-1.29(m,2H),0.93(t,J＝7.1Hz,3H).13C NMR(100MHz,DMSO-d6):169.55,163.38,162.81,140.26,131.54,130.73,129.10,128.20,126.25,123.82,122.17,119.12,117.30,39.25,29.65,19.81,13.71.HRMS(ESI):calculated for C17H17N2O3 +(M+H+)297.1234,found 297.1239.
NR-FAP: a pair of rotamers.1H NMR(400MHz,DMSO-d6):10.80(s,0.6H),10.66(d,J＝10.8Hz,0.4H),10.54(s,0.4H),9.06(d,J＝10.7Hz,0.4H),8.62(d,J＝7.8Hz,1H),8.41(s,0.6H),8.15(d,J＝7.4Hz,1H),7.92-7.80(m,3.6H),7.50(ddd,J＝12.9,8.7,2.1Hz,1H),6.47-6.40(m,1H).HRMS(ESI):calculated for C17H11N2O3 +(M+H+)291.0764,found291.0758.
(1) Three probe pairs, ONOO, prepared in example 1-Fluorescence response experiment of (2):
probe (stock solution 1.0mM, 200. mu.M), buffered with PBS (pH 7.4), diluted with water and different concentrations of ONOO were added-As a result of setting the final concentration of the probe at 2.0. mu.M and the concentration of PBS at 50mM, as shown in FIG. 1, when the probe BT-FAP was excited at 385nm, there was substantially no fluorescence emission at about 500nm, and it was found that there was no fluorescence emission at about 500nm, and that there was no fluorescence emission at different concentrations of ONOO-(ONOO corresponding in sequence from bottom to top-0. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M) is incubated, the fluorescence is significantly enhanced; when the probe NA-FAP is excited by 435nm, the probe basically has no fluorescence emission at about 560nm and has different concentrations of ONOO-(in sequence from bottom to topCorresponding ONOO -0. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M) is incubated, the fluorescence is significantly enhanced; when the probe NR-FAP is excited at 530nm, the probe basically has no fluorescence emission at about 620nm and has different concentrations of ONOO-(ONOO corresponding in sequence from bottom to top-At a concentration of 0. mu.M, 0.5. mu.M, 1. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, 3. mu.M, 3.5. mu.M, 4. mu.M) was significantly enhanced. And the fluorescence intensity and ONOO of the three probes are respectively 500nm, 560nm and 620nm-The concentrations showed a good linear correlation (fig. 2). The detection limits are respectively calculated to be 64nM, 28nM and 102nM according to the formula 3 sigma/k, which indicates that the three probes can detect the endogenous trace amount of ONOO in the biological system-The ability of the cell to perform.
(2) Three probe pairs, ONOO, prepared in example 1-Kinetic response experiment of (2):
on the basis of the above (1), the invention further researches three probe pairs ONOO-The results are shown in FIG. 3, and the three probes (2.0. mu.M) themselves are relatively weak in fluorescence at 500nm, 560nm and 620nm, respectively, when ONOO is added-(10. mu.M for BT-FAP, 10. mu.M for NA-FAP and 4. mu.M for NR-FAP), the fluorescence rapidly increases and reaches the peak within 4s, indicating that the probe can instantaneously respond to ONOO-Can capture the ONOO with high reactivity and short half-life period in the physiological environment-。
(3) Three probe selectivity experiments prepared in example 1:
on the basis of the step (1), the invention further carries out three probe pairs of ONOO-The results of the selectivity study are shown in FIG. 4, reference numeral 1 denotes ONOO-(test BT-FAP at 10. mu.M, test NA-FAP at 10. mu.M, and test NR-FAP at 4. mu.M), 2 for blank, 3 for 100. mu.M ClO-And 4 represents 100. mu.MH2O2And 5 represents 100. mu.M·OH, 6 represents 100. mu. MO2 ·-And 7 represents 100. mu.M1O28 for 100. mu.M NO, 9 for 100. mu.M TBHP, 10 for 5mM GSH, 11 for 100. mu. MHcy, 12 for 5mMCys, 13 for 100. mu.M vitamin C, 14 for 100. mu. M H2S, 15 represents 100. mu.M HSO3 -And 16 represents 100. mu. MSO 4 2-17 represents 100. mu.M PO 4 3-18 represents 100. mu. MNO2 -And 19 represents 100. mu.M NO3 -And 20 represents 100. mu. MAcO -21 represents 100. mu.M HCO3 -And 22 represents 100. mu.M CO 3 2-23 represents 100. mu.M Na+And 24 represents 100. mu. M K+And 25 represents 100. mu.M Mg 2+26 stands for 100. mu.M Cu 2+27 represents 100. mu.M Zn2+And 28 represents 100. mu. MCa2+And 29 represents 100. mu. MFe2+And 30 represents 100. mu. MFe3+. As can be seen from FIG. 4, none of the other potentially interfering substances was able to cause a change in fluorescence of the probe, including reactive oxygen species/reactive nitrogen species (H)2O2，TBHP，NO，1O2，O2 ·-，ClO-，·OH), active sulfur (GSH, Cys, Hcy, H)2S), anion (HSO)3 -，SO4 2-，PO4 3-，NO2 -，NO3 -，AcO-，HCO3 -And CO3 2-) Cation (Na)+，K+，Mg2+，Cu2+，Zn2+，Ca2+，Fe2+And Fe3+) And vitamin C. In contrast, all three probes were on ONOO alone-(corresponding to reference numeral 1) shows a significant fluorescence enhancement in the presence. In conclusion, the probes BT-FAP, NA-FAP and NR-FAP prepared in the embodiment are paired with ONOO-Has high specificity, and can be used in physiological environment-Fluorescence visualization study of (2).
(4) Probe-to-live cell staining imaging experiment:
as shown in FIG. 5, the present invention further verifies that the three probes BT-FAP, NA-FAP and NR-FAP prepared in example 1 can realize the on-OO on the cellular level-Detection of (3). Control group: FIGS. a, d and g are staining patterns of probes BT-FAP, NA-FAP and NR-FAP, respectively, on HepG2 cells. Experimental groups: PanG 2 cells in panels b, e and h were first treated with ONOO-Incubating release agent SIN-1 for 30min, thenAnd then, respectively incubating for 20min by using the three probes, and then carrying out confocal fluorescence imaging. Clearing group: in panels c, f and i, uric acid (a known ONOO) is first used-Scavenger) pre-treatment of HepG2 cells for 3 hours followed by ONOO-And (3) incubating the release agent SIN-1 for 30min, then incubating for 20min by using the three probes respectively, and finally performing confocal fluorescence imaging. The results show that the fluorescence intensity of the cells in the experimental group is obviously increased compared with the fluorescence intensity of the cells in the control group, and the fluorescence intensity of the cells is obviously reduced compared with the fluorescence intensity of the cells in the experimental group after the cells are pretreated by the uric acid. Thus, the probes BT-FAP, NA-FAP and NR-FAP can be used for detecting ONOO in living cells-Fluctuation of the content.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (9)
1. The fluorescent probe for detecting peroxynitrite based on the formamide recognition group is characterized in that: BT-FAP, NA-FAP or NR-FAP, respectively, wherein:
the structural formula of the BT-FAP is as follows:
or; the structural formula of the NA-FAP is as follows:
or; the structural formula of the NR-FAP is as follows:
2. the method for preparing the formamide recognition group-based fluorescent probe for detecting peroxynitrite according to claim 1, wherein the method comprises the following steps:
the reaction equations are respectively:
the reaction steps are as follows: respectively dissolving the compound 1, 2 or 3 in the reaction equation in formic acid, uniformly stirring to obtain a mixed solution, and reacting for a set time to obtain the compound.
3. The method of claim 2, wherein: the ratio of the compound 1, 2 or 3 to formic acid is: 0.2-1.5 mM: 0.5-5 mL.
4. The method of claim 2, wherein: the reaction temperature is 60-100 ℃, and the reaction time is 3-6 h.
5. The method of claim 2, wherein: the reaction temperature is 75-85 ℃, and the reaction time is 4.5-5 h.
6. The production method according to any one of claims 2 to 5, wherein: also comprises the steps of spin-drying and purifying the reaction product solution.
7. The method of claim 6, wherein: purifying the reaction product by silica gel column chromatography.
8. The method of claim 7, wherein: the eluent of the silica gel column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 80-130:1, 75-125:1 and 40-60:1 in sequence.
9. The method of claim 7, wherein: the eluent of the silica gel column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol used for purifying three reaction products of the compound 1, 2 or 3 and formic acid are respectively 100:1, 100:1 and 50:1 in sequence.
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