Golgi apparatus targeting calcium ion fluorescent probe, preparation method and application

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a Golgi apparatus targeting calcium ion fluorescent probe, a preparation method and application. The probe disclosed by the invention is simple to synthesize and relatively high in yield; according to the invention, specific and rapid detection of calcium ions in an aqueous solution is realized; according to the invention, the detection of calcium ions in the living cell Golgi apparatus is realized.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a Golgi-targeted calcium ion fluorescent probe, its preparation method, and its application. Background Technology

[0002] The Golgi apparatus is the center of the vesicle transport network in eukaryotic cells and a central organelle for the processing, sorting, and secretion of lipids and glycoproteins. Over the past two decades, with the help of gene-coded or chemiluminescent probes, a relatively systematic understanding of calcium signaling in the cytoplasm, mitochondria, endoplasmic reticulum, and even lysosomes has been established. However, the presence of calcium within the Golgi apparatus lumen remains a concern. 2+ The dynamic regulation of concentration and its relationship with physiological and pathological processes remain in the "dark zone".

[0003] Ca in the Golgi body cavity 2+ The concentration is approximately 300-600 μM, much higher than that in the cytoplasm (~100 nM). This gradient is caused by SPCA1 (Secretory Pathway Ca). 2+ The ion gradient is maintained by ATPase 1 and PMR1 plasma pumps and finely regulated by ion channels and exchangers. This gradient not only drives the optimal conformation of intraluminal enzymes such as mannosidase and glycosyltransferase, but also determines the formation and budding efficiency of COPII / COPI vesicles. Traditional methods (such as electron microscopy X-ray microanalysis and subcellular separation combined with atomic absorption) can only provide static or averaged data and cannot capture millisecond-level calcium transients, leaving the core question of "how the gradient is dynamically regulated" unresolved. In addition, Golgi body calcium imbalance is directly related to major diseases. In Alzheimer's disease, abnormal retention of β-secretase (BACE1) in the Golgi apparatus is significantly associated with decreased luminal calcium; during tumor metastasis, SPCA1 overexpression induces increased Golgi body calcium, promoting the maturation of glycosylation-dependent adhesion molecules; in diabetic pancreatic β cells, Golgi body calcium depletion leads to misfolding of proinsulin. Without real-time visualization of this organelle's calcium signaling, it is difficult to clarify the molecular switches in the early stages of disease, let alone develop precise intervention strategies.

[0004] While commercially available probes like Rhod-2 and Fluo-4 can be chemically modified to introduce Golgi targeting sequences (such as the transmembrane domain of TGN38), they suffer from issues like transmembrane leakage, endosome mislocalization, and low signal-to-noise ratio. Gene-encoded probes like GCaMP6f fused with Golgi resident proteins (such as GalT and GPP130) can partially address localization problems, but their pH sensitivity and low brightness make subcellular quantification difficult. Therefore, exploring convenient, inexpensive methods for qualitative and quantitative detection of calcium ions, along with in-situ visual monitoring of biological samples, is crucial. When an organic fluorescent probe reacts with a specific target, its fluorescence signal changes, achieving the detection purpose. Fluorescence analysis methods offer advantages such as low limits of emission, high sensitivity, good selectivity, small sample sizes, and rapid, simple methods. Furthermore, they allow for non-invasive imaging detection of intracellular target molecules, enabling real-time, online observation of signal changes. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a Golgi-targeted calcium ion fluorescent probe, its preparation method and application, so as to improve the detection effect.

[0006] This invention provides a Golgi-targeted calcium ion fluorescent probe, the structural formula of which is: Its molecular formula is as follows: C 38 H 57 N3O7.

[0007] This invention provides a method for preparing a Golgi-targeted calcium ion fluorescent probe, comprising the following steps: 1) 2-(2-aminoethyl)-8-bromo-1H-benzo[de]isoquinoline-1,3(2H)-dione, tetradecanoic acid, benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate, and triethylamine were mixed, reacted, and purified to obtain compound 2; the structural formula of compound 2 is: ; 2) Compound 2, aza-15-crown ether-5, N,N-dimethylformamide, and carbonate were mixed and reacted at 110-130°C. After purification, a targeted calcium ion fluorescent probe was obtained.

[0008] Preferably, step 1) also includes the solvent dichloromethane.

[0009] Preferably, the carbonate is potassium carbonate.

[0010] Preferably, in step 1), after the reaction is complete, dichloromethane in the reaction solution is removed by vacuum distillation using a rotary evaporator.

[0011] Preferably, in step 1), the purification process is performed using a silica gel chromatography column.

[0012] Preferably, in step 2), the purification process is performed using a silica gel chromatography column.

[0013] This invention provides an application of the Golgi apparatus-targeted calcium ion fluorescent probe, which is used to detect calcium ions in a sample. The sample is an aqueous solution or a biological sample.

[0014] The detection method can be: Observe the changes in the fluorescence spectrum of the water environment before and after the addition of the calcium fluorescent probe; the fluorescence excitation wavelength is 420 nm. Alternatively, under 365 nm light source illumination, observe the fluorescence changes of the water environment before and after adding the calcium ion fluorescent probe with the naked eye; Alternatively, observe the changes in fluorescence imaging of the biological environment before and after the addition of the Golgi calcium ion fluorescent probe.

[0015] The change in the fluorescence spectrum refers to the change in the fluorescence peak at 550 nm; if the peak at 550 nm becomes larger, it indicates the presence of calcium ions.

[0016] The fluorescence change refers to the change in fluorescence from colorless to yellow under 365 nm light source illumination. The change in the fluorescence imaging pattern refers to the enhanced fluorescence in the green channel during live-cell imaging excited by a 420 nm light source.

[0017] The specific testing method is as follows: 1) Dissolve probe 3 in DMSO to prepare probe stock solution; 2) Add the probe stock solution to the test solution; The fluorescence spectrum of the test solution was tested using a fluorescence spectrometer. The change in the fluorescence peak at 550 nm was observed. If the peak at 550 nm became larger, it indicated the presence of calcium ions. The excitation wavelength of the fluorescence spectrometer was 420 nm. Alternatively, if the fluorescence of the test solution changes from colorless to yellow under 365 nm light source illumination, it indicates the presence of calcium ions. 3) Add the probe stock solution to the biological sample, use a confocal microscope and a light source with an excitation wavelength of 405 nm to excite the sample, and collect the fluorescence of the blue channel; if the fluorescence of the blue channel is enhanced, it indicates that calcium ions are present in the Golgi apparatus.

[0018] The beneficial effects of this invention are that the probe of this invention is simple to synthesize and has a high yield; this invention achieves specific and rapid detection of calcium ions in aqueous solution; this invention achieves the detection of calcium ions in the Golgi apparatus of living cells.

[0019] For the detection of calcium ions, this invention firstly recognizes that calcium ions in aqueous solution can cause changes in the fluorescence spectrum of a fluorescent probe. Therefore, the content of calcium ions in the solution can be determined by observing the degree of spectral change in a fluorescence spectrometer, thus enabling quantitative detection. Secondly, fluorescence imaging of live cells incubated with the fluorescent probe and calcium ions is performed using a confocal microscope to observe changes in the fluorescence signal of the yellow channel, thereby achieving the purpose of ratio detection of calcium ions in the biological environment. Attached Figure Description

[0020] Figure 1 This is the 1H NMR spectrum of probe 3 in Example 1.

[0021] Figure 2 This is the mass spectrum of probe 3 in Example 1.

[0022] Figure 3 This shows the changes in the fluorescence spectrum of probe 3 (10 μmol / L) in PBS buffer solution (concentration 25 mmol / L, pH 7.4, containing 20% ​​DMSO) with the addition of different amounts of calcium ions in Example 2; The figure shows, from bottom to top, the fluorescence spectra of calcium ion concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 100 μmol / L.

[0023] Figure 4 The changes observed by the naked eye in Example 2 for probe 3 (10 μmol / L) in PBS buffer solution (concentration 25 mmol / L, pH 7.4, containing 20% ​​DMSO) and 10 equivalents of calcium ions under the illumination of a portable UV lamp at 365 nm are described.

[0024] Figure 5 This is a graph showing the change in fluorescence intensity at 550 nm of probe 3 (10 μmol / L) in PBS buffer solution (concentration 25 mmol / L, pH 7.4, containing 20% ​​DMSO) and 10 equivalents of calcium ions over time in Example 3.

[0025] Figure 6 This is a columnar fluorescence data chart showing the selectivity of probe 3 (10 μmol / L) in PBS buffer solution (concentration 25 mmol / L, pH 7.4, containing 33% DMSO) against different interfering analytes (10 μmol / L) in Example 4. In the diagram, 1.blank, 2.Al 3+ 3.Ba 2+ , 4.Fe 2+ 5.Cr 2+ 6.Cd 2+ 7.Hg2+ 8.Co 2+ 9.K + 10.Mg 2+ ,11.Mn 2+ ,12.Na + ,13.Ni 2+ 14.Pb 2+ ,15.Fe 3+ ,16.Cu 2+ 17.Zn 2+ .

[0026] Figure 7 This is a fluorescence data graph of probe 3 (10 μmol / L) in PBS buffer solution (concentration 25 mmol / L, pH 7.4, containing 20% ​​DMSO) and 10 equivalents of calcium ions at different pH values.

[0027] Figure 8 This is a fluorescence imaging diagram of the calcium ion response of probe 3 in HeLa cells in Example 5; In the figure, (A) is the image after adding probe 3 and incubating for 30 min, (B) is the image after adding 10 μM calcium ions and incubating with probe 3 for 30 min, (C) is the image after adding 10 μM calcium ions and incubating with probe 3 for 30 min, and (D) is the image after adding 10 μM calcium ions and 10 μM EDTA and incubating with probe 3 for 30 min; the excitation wavelength is 405 nm.

[0028] Figure 9 This is a fluorescence imaging image of the co-localization of probe 3 with a commercial Golgi apparatus probe after the calcium ion response in HeLa cells, as shown in Example 5.

[0029] The first column shows green channel imaging with an excitation wavelength of 405 nm; the second column shows red channel imaging using a commercially available Golgi targeting probe with an excitation wavelength of 561 nm; the third column shows bright field imaging of cells; and the fourth column shows the ratio of green channel imaging to red channel imaging. Detailed Implementation

[0030] Example 1 A method for preparing a Golgi-targeted calcium ion fluorescent probe includes the following steps: 1) 318 mg (1 mmol) of 2-(2-aminoethyl)-8-bromo-1H-benzo[de]isoquinoline-1,3(2H)-dione (compound 1) was placed in a round-bottom flask, followed by the addition of 228 mg (1 mmol) of tetradecanoic acid and 780 mg (1.5 mmol) of benzotriazol-1-yl-oxytripyrrolidinephosphide (PyBop). The mixture was dissolved in 15 mL of dichloromethane, and 10 mg (0.01 mmol) of triethylamine was added. The mixture was stirred at room temperature for 6 hours. Then, the dichloromethane in the reaction solution was removed by vacuum distillation using a rotary evaporator. The mixture was then purified by silica gel chromatography to obtain compound 2.

[0031] 2) 264 mg (0.5 mmol) of compound 2 was placed in a round-bottom flask, followed by the addition of 440 mg (2 mmol) of aza-15-crown ether-5, which was dissolved in 5 mL of N,N-dimethylformamide. Then, 105 mg (0.75 mmol) of potassium carbonate was added, and the mixture was stirred at 120 °C for 12 hours to remove N,N-dimethylformamide. The mixture was then purified by silica gel chromatography to obtain the targeted calcium ion fluorescent probe, namely probe 3 (compound 3).

[0032] The reaction route is as follows:

[0033] probe 3 1 The H NMR and mass spectra are as follows: Figure 1-2 As shown.

[0034] Example 2 Fluorescence spectral changes of probe 3 reacting with different equivalents of calcium ions The probe 3 prepared in Example 1 was dissolved in DMSO to prepare a probe stock solution with a concentration of 1 mmol / L (the concentration of probe 3 was 1 mmol / L); calcium chloride was dissolved in DMSO to prepare a stock solution with a concentration of 10 mmol / L, and solutions of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 100 μmol / L were prepared to obtain calcium ion working solutions.

[0035] Take 30 μL from the probe stock solution and add it to a 5 mL centrifuge tube. Add different equivalents (0-10 eq) of calcium ion stock solution (the equivalent refers to the multiple of the number of moles of calcium chloride in the calcium ion stock solution relative to the number of moles of probe in the probe stock solution), and different volumes of PBS aqueous solution (concentration 25 mmol / L, pH 7.4). Make up to 3 mL with DMSO to prepare a test solution with a probe concentration of 10 μmol / L and containing 20% ​​DMSO.

[0036] The fluorescence spectral changes of probe 3 with different equivalents of calcium ion reaction solutions were tested using a fluorescence spectrometer (excitation wavelength 420 nm). The fluorescence spectral changes are as follows: Figure 3 As shown.

[0037] Depend on Figure 3 It can be seen that as the amount of calcium chloride added gradually increases, the fluorescence peak of probe 3 solution at 550 nm gradually increases.

[0038] like Figure 4 As shown, under the illumination of a portable ultraviolet lamp (365 nm light source), the fluorescence changes from colorless to blue.

[0039] Example 3 Fluorescence changes of probe 3 and calcium ions over time Take 30 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Add 30 μL of calcium chloride stock solution with a concentration of 10 mmol / L, and then dilute it to 3 mL with 0.540 mL of DMSO and 2.400 mL of PBS aqueous solution (concentration 25 mmol / L, pH 7.4) to prepare a test solution with a probe concentration of 10 μmol / L, a calcium chloride concentration of 100 μmol / L, and containing 20% ​​DMSO.

[0040] The fluorescence spectrum of the sample was measured over time using an excitation wavelength of 370 nm.

[0041] Depend on Figure 5 It can be seen that the fluorescence at 550 nm reaches the response value almost instantaneously, and the fluorescence intensity at 550 nm remains basically unchanged as time increases.

[0042] Example 4 Selectivity study of probe 3 for different interfering analytes Take 30 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Add 30 μL of each of the following analytes at a concentration of 1.00 mM: 1. blank, 2. Al. 3+ 3. Ba 2+ 4. Fe 2+ 5. Cr 2+ 6. Cd 2+ 7. Hg 2+ 8. Co 2 + 9. K + 10. Mg 2+ 11. Mn 2+ 12. Na + 13. Ni 2+ 14. Pb2+ 15. Fe 3+ 16. Cu 2+ 17. Zn 2+ The sample was diluted to 3 mL with 0.940 mL DMSO and 2.000 mL PBS aqueous solution (concentration 25 mmol / L, pH 7.4) to prepare a test solution with a probe concentration of 10 μmol / L containing 33% DMSO. The fluorescence spectrum change of the test solution was detected after 5 minutes of reaction.

[0043] Depend on Figure 6 It was observed that the fluorescence intensity of the test solutions for all analytes did not change significantly compared to the blank test solution. However, the fluorescence intensity of the test solution with added calcium ions was significantly enhanced. These results indicate that probe 3 exhibits good selectivity for calcium ions.

[0044] Test study on the response of probe 3 at different pH values Take 30 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Dilute it to 3 mL with 0.970 mL DMSO and 2.000 mL PBS aqueous solution (concentration 25 mmol / L, pH values ​​4, 5, 6, 7, 7.4, 8, 9, 10, 11) to prepare a test solution with a probe concentration of 10 μmol / L and containing 33% DMSO. Detect the change in fluorescence spectrum of the test solution after 5 minutes of reaction.

[0045] Depend on Figure 7 It can be observed that the probe blank test solution maintains weak fluorescence at different pH values, while the strong response to calcium ions is observed in the pH range of 7-11.

[0046] Example 5 Fluorescence imaging study of probe 3 and calcium ions in cells 10 μL of the probe stock solution from Example 2 was added to a culture dish containing H9c2 cells (containing 1 mL of DMEM medium), with a probe concentration of 10 μmol / L. The cells were incubated for 30 minutes as the control group. Samples in experimental group 1 were incubated with 10 μM calcium chloride for 30 minutes beforehand to increase intracellular calcium ions. Samples in experimental group 2 were incubated with 20 μM calcium ions for 0.5 h beforehand, and samples in experimental group 3 were incubated with 20 μM calcium ions + 10 μM EDTA for 0.5 h beforehand. Subsequently, fluorescence imaging of the control and experimental groups was performed using a confocal microscope. A light source with an excitation wavelength of 405 nm was used to excite the cells, and the fluorescence in the green channel was collected. The results are as follows: Figure 8 As shown.

[0047] In the fluorescence imaging of the control group, green fluorescence could be observed; however, in experimental group 1, obvious green channel fluorescence could be observed, in experimental group 2, relatively weak green fluorescence could be observed, and in experimental group 3, extremely weak green fluorescence could be observed.

[0048] Experimental results show that probe 3 can detect calcium ions in the cellular environment through confocal microscopy, and has potential practical application value.

[0049] Example 6 10 μL of the probe stock solution from Example 2 was added to a culture dish containing H9c2 cells (containing 1 mL of PBS medium), with a probe concentration of 10 μmol / L. The cells were incubated for 30 minutes as the control group. Samples in experimental group 1 were incubated with calcium ions to increase intracellular calcium levels, and the Golgi apparatus of the cells was stained using Beyotime's commercial Golgi targeting fluorescent probe (red). After staining, the cells were incubated with probe 3 working solution for 30 minutes. Subsequently, fluorescence imaging of the control and experimental groups was performed using a confocal microscope. A light source with an excitation wavelength of 405 nm was used to excite the cells, and fluorescence in the green and blue channels was collected. The results are as follows: Figure 9 As shown.

[0050] As can be seen, the green and blue co-localization of the Golgi apparatus in the experimental group was good. The experimental results indicate that probe 3 can be effectively targeted to the Golgi apparatus, demonstrating potential practical application value.

[0051] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0052] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A Golgi apparatus-targeted calcium ion fluorescent probe, characterized in that, The structural formula of the fluorescent probe is: 。 2. A method for preparing the Golgi-targeted calcium ion fluorescent probe as described in claim 1, characterized in that, Includes the following steps: 1) 2-(2-aminoethyl)-8-bromo-1H-benzo[de]isoquinoline-1,3(2H)-dione, tetradecanoic acid, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, and triethylamine were mixed, reacted, and purified to obtain compound 2; the structural formula of compound 2 is: ； 2) Compound 2, aza-15-crown ether-5, N,N-dimethylformamide, and carbonate were mixed and reacted at 110-130℃. After purification, a targeted calcium ion fluorescent probe was obtained.

3. The preparation method according to claim 2, characterized in that, Step 1) also includes the solvent dichloromethane.

4. The preparation method according to claim 2, characterized in that, The carbonate is potassium carbonate.

5. The preparation method according to claim 3, characterized in that, In step 1), after the reaction is complete, dichloromethane in the reaction solution is removed by vacuum distillation using a rotary evaporator.

6. The preparation method according to claim 2, characterized in that, In step 1), the purification process is performed using a silica gel chromatography column.

7. The preparation method according to claim 2, characterized in that, In step 2), the purification process is performed using a silica gel chromatography column.

8. The application of the Golgi apparatus-targeted calcium ion fluorescent probe as described in claim 1, characterized in that, The Golgi-targeted calcium ion fluorescent probe is used to detect calcium ions in the sample.

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