CN114621172B - Golgi apparatus targeting ferrous ion fluorescent probe and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a high-efficiency matrix targeting ferrous ion fluorescent probe and a preparation method and application thereof, and belongs to the technical field of ferrous ion probes. The molecular formula of the probe is as follows: c (C) 27 H 42 N 2 O 4 Has the following structural formula:the invention provides a fluorescence analysis method with high accuracy, which can detect ferrous ions in a golgi apparatus in water environment and in-vitro living cell environment. After the probe responds to ferrous ions, the fluorescence intensity at 460nm is enhanced. The probe can also detect ferrous ions in the golgi in the living cells in vitro by a confocal fluorescence microscope, and perform fluorescence imaging. The probe of the invention has simple synthesis, higher yield and certain potential practical value.
Description
Technical Field
The invention belongs to the technical field of ferrous ion probes, and particularly relates to a golgi targeted ferrous ion fluorescent probe, a preparation method and application thereof.
Background
Iron is the most abundant transition metal on earth, and ferrous ions in the form of divalent ions have strong redox activity. Living organisms utilize the redox activity of ferrous iron to perform a number of physiologically essential processes such as oxygen transport, DNA synthesis, respiration and metabolic reactions. However, excess ferrous iron is potentially toxic due to its uncontrolled redox activity. Ferrous ions, defined as non-protein or weakly protein bound forms, induce oxidative damage to the body through the Fenton chemical reaction. Golgi, the central organelle of intracellular membrane binding, plays a key role in the transport, processing and sorting of newly synthesized membranes, secreted proteins and lipids.
The fluorescence analysis method has the characteristics of low limit, high sensitivity, good selectivity, small sampling amount, simple and quick method and the like, can carry out non-invasive imaging detection on target molecules in cells, can realize real-time online, and can observe signal change in a specific image. Therefore, it is necessary to provide a ferrous ion fluorescent probe capable of detecting signals rapidly and allowing signals to be observed easily. In addition, in view of the unique biological functions of the golgi apparatus, the synthesis of the golgi apparatus-targeted ferrous ion probe to obtain intermediate information is of great significance for research and understanding of golgi apparatus-related subjects.
Disclosure of Invention
The invention provides a golgi targeted ferrous ion fluorescent probe, a preparation method and application thereof, and is abbreviated as probe 3, which can realize the detection of ferrous ions in aqueous solution and in-vitro biological sample golgi.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a high-efficiency matrix targeting ferrous ion fluorescent probe has a molecular formula as follows: c (C) 27 H 42 N 2 O 4 Has the following structural formula:
the Golgi apparatus targeting ferrous ion probe of the invention can resist the interference of aluminum ion, barium ion, calcium ion, chromium ion, cadmium ion, mercury ion, cobalt ion, potassium ion, magnesium ion, manganese ion, sodium ion, nickel ion, lead ion, iron ion, copper ion and zinc ion.
A preparation method of a Golgi apparatus targeting ferrous ion fluorescent probe comprises the following steps:
(1) 100mg of 7- (dimethylamino) coumarin-4-acetic acid is placed in a container, 129mg of tetramine and 312mg of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate PyBop are added, dichloromethane is used for dissolving, 8 drops of triethylamine are added, stirring is carried out for 12 hours at normal temperature under the protection of nitrogen, dichloromethane in the reaction solution is removed, and the compound 2 is obtained through separation and purification by a silica gel chromatographic column, wherein the structural formula of the compound 2 is as follows:
the chemical reaction equation of the step is as follows:
(2) 22mg of compound 2 is weighed and placed in a container, dichloromethane is used for dissolution, 69mg of m-chloroperoxybenzoic acid which is dissolved in dichloromethane in advance is added by a constant pressure dropping funnel, the mixture is stirred for 0.5 hour under ice bath, the mixture is stirred for 5 hours at room temperature, after the reaction is finished, the dichloromethane is removed from the solution, and a target probe, namely a probe 3 is obtained through separation and purification of a silica gel chromatographic column, wherein the structural formula of the probe 3 is as follows:
the chemical reaction equation of the step is as follows:
the overall synthesis route of the probe of the invention is as follows:
further, the methylene dichloride in the reaction liquid can be removed by reduced pressure distillation through a rotary evaporator in the whole reaction process. The triethylamine is added dropwise.
The application of the Golgi apparatus targeting ferrous ion fluorescent probe is that the fluorescent probe is used for detecting ferrous ion in water environment, and also can be used for detecting ferrous ion in the Golgi apparatus in-vitro living cell environment, obtaining intermediate information and performing fluorescence imaging.
The above application specifically includes:
observing the change of fluorescence spectra of the water environment to be measured before and after the addition of the Golgi apparatus ferrous ion fluorescence probe; the fluorescence excitation wavelength is 370nm;
or observing the change of the fluorescence imaging diagram of the biological environment to be detected before and after the ferrous ion fluorescent probe is added.
The biological environment may be living cells ex vivo.
The change of the fluorescence spectrum refers to: a change in fluorescence peak at 460nm in the fluorescence spectrum; if the peak at 460nm is raised, then this indicates that the superoxide anion is contained. Preferably, the fluorescence spectrum is observed using a fluorescence spectrometer.
The fluorescence change refers to: under the irradiation of 370nm light source, the fluorescence is obviously enhanced.
The change of the fluorescence imaging diagram refers to: adding the probe mother liquor into a biological sample, exciting by using a confocal microscope and a light source with an excitation wavelength of 405nm, and collecting fluorescence of a blue channel; an increase in fluorescence of the blue channel was observed, indicating that the super-oxyanion was included. Preferably, a confocal microscope is used.
Further, the specific detection method comprises the following steps:
(1) Dissolving the probe in ethanol to prepare a probe mother solution;
(2) Adding the probe mother solution into the liquid to be detected;
testing the fluorescence spectrum of the liquid to be tested by using a fluorescence spectrometer, wherein the change of the fluorescence peak value at 460nm is shown to contain superoxide anions if the peak value at 460nm is larger; wherein the excitation wavelength of the fluorescence spectrometer is 370nm;
(3) Adding the probe mother liquor into a biological sample, exciting by using a confocal microscope and a light source with an excitation wavelength of 405nm, and collecting fluorescence of a blue channel; an increase in fluorescence of the blue channel was observed, indicating that the super-oxyanion was included.
First, ferrous ions in the aqueous solution can cause the fluorescence spectrum of the fluorescent probe to change, so that the content of ferrous ions in the solution can be judged by observing the change degree of the spectrum in the fluorescence spectrometer, thereby quantitatively detecting. And secondly, performing fluorescence imaging on living cells incubated with the fluorescent probe 3 and ferrous ions by using a confocal microscope, and observing the change of a fluorescence signal of a blue channel to achieve the purpose of detecting ferrous ions in a biological environment by using a ratio.
Advantageous effects
The invention has the advantages that: (1) the synthesis of the probe is simple, and the yield is high; (2) The invention realizes the specificity and rapid detection of ferrous ions in the aqueous solution; (3) The invention realizes the detection of ferrous ions in the in vitro living cell Golgi apparatus.
Drawings
FIG. 1 is a diagram of Compound 2 in example 1 1 H NMR spectrum;
FIG. 2 is a diagram of Compound 2 in example 1 13 C NMR spectrum;
FIG. 3 is a mass spectrum of compound 2 of example 2;
FIG. 4 is a diagram showing a probe 3 in example 2 1 H NMR spectrum;
FIG. 5 shows the probe 3 of example 2 13 C NMR spectrum;
FIG. 6 is a mass spectrum of probe 3 in example 2;
FIG. 7 is a graph showing the change of the fluorescence spectrum of the probe 3 according to the addition of different amounts of ferrous ions in example 2; in the figure, the ferrous ion concentration is 0, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150 and 170 mu mol/L fluorescence spectrum in sequence from bottom to top;
FIG. 8 is a graph showing the change in fluorescence intensity at 460nm with time of the change in the iron ions and the probe 3 in example 3;
FIG. 9 is a graph of competitive column fluorescence data for probe 3 of example 4 for different interfering analytes; in the figure, ferrous ion 1, aluminum ion 2, barium ion 3, calcium ion 4, chromium ion 5, cadmium ion 6, mercury ion 7, cobalt ion 8, potassium ion 9, magnesium ion 10, manganese ion 11, sodium ion 12, nickel ion 13, lead ion 14, iron ion 15, copper ion 16, zinc ion 17;
FIG. 10 is a graph showing fluorescence response of probe 3 in example 4 to different pH conditions;
FIG. 11 is a fluorescence imaging of probe 3 of example 5 in response to ferrous ions in HepG2 cells; in the figure, (a) is an image after incubation for 30min with the addition of probe 3, (B) is an image after incubation for 30min with probe 3 after pre-incubation for 30min with the addition of 2eq of ferrous ions, (C) is an image after incubation for 30min with probe 3 after pre-incubation for 30min with the addition of 5eq of ferrous ions, and (D) is an image after incubation for 30min with probe 3 after pre-incubation for 30min with the addition of 10eq of ferrous ions;
FIG. 12 is a co-localized fluorescence imaging of probe 3 of example 5 with a commercial Golgi probe after a response to ferrous ions in HepG2 cells. The first column images the blue channel; the second column is a ratio graph of green channel imaging to blue channel imaging of the commercial golgi targeting probe; the excitation wavelength was 405nm.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1
Synthesis of Compound 2:
100mg (0.4 mmol) of 7- (dimethylamino) coumarin-4-acetic acid was placed in a round bottom flask, 129mg (0.6 mmol) of decamine and 312mg (0.6 mmol) of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate were added, dissolved in methylene chloride, 8 drops of triethylamine were added, and stirring was carried out at room temperature overnight to complete the reaction. After the completion of the reaction, the solvent (methylene chloride) was removed by distillation under reduced pressure by a rotary evaporator to give a crude product. Purification by silica gel (100-200 mesh) column chromatography using dichloromethane as eluent gave 200mg (90% yield) of white solid. 1 H NMR(500MHz,CDCl3)δ7.48(d,J＝9.0Hz,1H),6.61(dd,J＝9.0,2.5Hz,1H),6.49(d,J＝2.5Hz,1H),6.03(s,1H),5.76(d,J＝4.8Hz,1H),3.61(s,2H),3.20(dd,J＝13.2,6.9Hz,2H),3.05(s,6H),1.46–1.37(m,2H),1.22(d,J＝26.9Hz,23H),0.88(t,J＝6.9Hz,3H). 13 C NMR(126MHz,CDCl3)δ167.83,161.67,156.06,153.12,149.91,125.76,110.01,109.19,108.16,97.96,40.67,40.14,31.86,29.71,29.39,26.82,22.48,14.12.m/z[M+H] + calcd.for 443.3268,found 443.3282.
Synthesis of probe 3:
22mg (0.05 mmol) of Compound 2 was weighed, dissolved in methylene chloride, 69mg (0.4 mmol) of m-chloroperoxybenzoic acid dissolved in anhydrous methylene chloride for extraction was added dropwise under ice bath conditions at constant pressure, stirred for 0.5 hour under ice bath conditions, stirred at room temperature for 5 hours, and the reaction was completed. After the completion of the reaction, the solvent (methylene chloride) was removed by distillation under reduced pressure using a rotary evaporator to give probe 3, which was prepared by using methylene chloride: ethanol=50: 2 as eluent, silica gel (100-200 mesh) column chromatography gave 9mg of white solid (40% yield). 1 H NMR(500MHz,CDCl3)δ7.48(d,J＝9.0Hz,1H),6.61(dd,J＝9.0,2.5Hz,1H),6.49(d,J＝2.5Hz,1H),6.03(s,1H),5.76(d,J＝4.8Hz,1H),3.61(s,2H),3.20(dd,J＝13.2,6.9Hz,2H),3.05(s,6H),1.46–1.37(m,2H),1.22(d,J＝26.9Hz,23H),0.88(t,J＝6.9Hz,3H). 13 C NMR(126MHz,CDCl3)δ167.13,159.65,156.91,153.56,149.06,126.33,119.60,117.57,116.23,109.58,77.29,77.04,76.78,63.24,40.13,40.03,31.92,29.69,29.66,29.61,29.58,29.49,29.35,29.29,26.98,22.68,14.11。m/z[M+H] + calcd.for 459.3217,found 459.3217.
Example 2
Fluorescence spectrum variation of Probe 3 reaction with different equivalent of ferrous chloride
Probe 3 prepared in example 1 was dissolved in DMSO to prepare a probe stock solution having a concentration of 1mmol/L (probe 3 concentration of 1 mmol/L); ferrous chloride was formulated with ethanol as 0, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150, and 170 μmol/L solutions. 30. Mu.L of the mother liquor of the probe was taken out and added to a 5mL centrifuge tube, and a different equivalent (0-17 eq) of the mother liquor of ferrous chloride (the equivalent means a multiple of the number of moles of ferrous chloride in the mother liquor of ferrous chloride relative to the number of moles of probe in the mother liquor of the probe) was added, and diluted to 3mL with 0.150mL of ethanol and a different volume of aqueous PBS (concentration 25mmol/L, pH 7.4) to prepare a test solution containing 5% ethanol at a probe concentration of 10. Mu. Mol/L. The fluorescence spectrum change (excitation wavelength is 370 nm) of the probe and the ferrous ion reaction liquid with different equivalent weights is tested by a fluorescence spectrometer, and the fluorescence spectrum change condition is shown in figure 7. As can be seen from fig. 7, the fluorescence peak of the probe 3 solution at 460nm gradually increased as the amount of ferrous ions added gradually increased.
Example 3
Fluorescence change of probe 3 and ferrous ion with time
From the fluorescent probe mother liquor of example 2, 30. Mu.L was taken out and added to a 5mL centrifuge tube, and 30. Mu.L of a ferrous ion mother liquor having a concentration of 10mmol/L was added, and then 0.150mL of ethanol and 2.850mL of an aqueous PBS solution (having a concentration of 25mmol/L, pH 7.4) were diluted to 3mL to prepare a test solution having a probe concentration of 10. Mu. Mol/L, a ferrous ion concentration of 10mmol/L and 5% ethanol. The fluorescence spectrum over time was measured with an excitation wavelength of 370 nm. As can be seen from FIG. 8, the fluorescence at 460nm reaches a response value at 5min, and the fluorescence intensity at 460nm is substantially unchanged as time increases.
Example 4
Selective investigation of Probe 3 for different interfering analytes
30. Mu.L of the fluorescent probe mother liquor from example 2 was taken into a 5mL centrifuge tube, and analytes at a concentration of 1.00mM were added, respectively: aluminum ion, barium ion, calcium ion, chromium ion, cadmium ion, mercury ion, cobalt ion, potassium ion, magnesium ion, manganese ion, sodium ion, nickel ion, lead ion, iron ion, copper ion, zinc ion. The sample was diluted to 3mL with 0.150mL of ethanol and 2.850mL of aqueous PBS (25 mmol/L, pH 7.4) to prepare a test solution containing 5% ethanol at a probe concentration of 10. Mu. Mol/L. After 5 minutes of reaction, the change in fluorescence spectrum of the test solution was detected. From fig. 9, it can be seen that the fluorescence intensity of the test solution for each analyte did not significantly change from that of the blank test solution. However, the fluorescence intensity of the test solution to which the ferrous ions are added is significantly enhanced. The experimental results show that the probe 3 has good selectivity for ferrous ions. FIG. 10 is a graph showing the fluorescence response of probe 3 (10. Mu.M) in PBS buffer (25 mmol/L, pH 7.4, containing 5% ethanol) to different pH.
Example 5
Fluorescence imaging study of Probe 3 and ferrous ions in cells
10. Mu.L of the fluorescent probe mother liquor in example 2 was taken out and added to a culture dish (containing 1mL of DMEM medium) in which HepG2 cells were grown, and the probe concentration was 10. Mu. Mol/L, and incubated for 30 minutes as a control group; samples of the experimental group were incubated with 20, 50, 100. Mu. Mol of ferrous ion solution for 30min in advance to excite intracellular ferrous ions, then fluorescence imaging was performed on the control group and the experimental group with a confocal microscope, excitation was performed using a light source with an excitation wavelength of 405nm, and fluorescence of the blue channel was collected, and the results are shown in FIG. 11. In fluorescence imaging of the control group, blue fluorescence can be observed; however, in the experimental group 1, blue channel fluorescence was observed, and in the experimental group 2, relatively strong blue fluorescence was observed, and in the experimental group 3, extremely strong blue fluorescence was observed. The experimental result shows that the probe 3 can detect ferrous ions in the cell environment through a confocal microscope, and has potential practical application value.
10. Mu.L of the fluorescent probe mother liquor in example 2 was taken out and added to a culture dish (containing 1mL of PBS medium) in which HepG2 cells were grown, and the probe concentration was 10. Mu. Mol/L, and incubated for 30 minutes as a control group; samples of experimental group 1 were incubated with a ferrous ion solution of 100. Mu. Mol/L for 30min in advance to excite intracellular ferrous ions, and the cell golgi apparatus was stained with a Biyun-Tian commercial golgi apparatus-targeted fluorescent probe (green), and cells were incubated with probe 3 working solution for 30min after staining. Subsequently, fluorescence imaging was performed on the control group and the experimental group by using a confocal microscope, respectively, and fluorescence was collected in the green and blue channels by excitation using a light source having an excitation wavelength of 405nm, and the results are shown in fig. 12. It can be seen that the experimental group golgi green and blue co-localization is good. Experimental results show that the probe 3 can be well targeted to the Golgi apparatus, and has potential practical application value.
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is evident that all other embodiments obtained by a person skilled in the art without making any inventive effort, based on the above-described embodiments of the invention, shall fall within the scope of protection of the invention.
Claims (1)
1. The high-efficiency matrix targeting ferrous ion fluorescent probe is characterized by comprising the following molecular formula: c (C) 27 H 42 N 2 O 4 Has the following structural formula:
the preparation method of the fluorescent probe comprises the following steps:
(1) 100mg of 7- (dimethylamino) coumarin-4-acetic acid is placed in a container, 129mg of tetramine and 312mg of benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate PyBop are added, dichloromethane is used for dissolving, 8 drops of triethylamine are added, stirring is carried out for 12 hours at normal temperature under the protection of nitrogen, dichloromethane in the reaction solution is removed, and the compound 2 is obtained through separation and purification by a silica gel chromatographic column, wherein the structural formula of the compound 2 is as follows:
the chemical reaction equation of the step is as follows:
(2) 22mg of compound 2 is weighed and placed in a container, dichloromethane is used for dissolution, 69mg of m-chloroperoxybenzoic acid which is dissolved in dichloromethane in advance is added by a constant pressure dropping funnel, the mixture is stirred for 0.5 hour under ice bath, the mixture is stirred for 5 hours at room temperature, after the reaction is finished, the dichloromethane is removed from the solution, and a target probe, namely a probe 3 is obtained through separation and purification of a silica gel chromatographic column, wherein the structural formula of the probe 3 is as follows:
the chemical reaction equation of the step is as follows:
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