Near-infrared ratio type fluorescent probe for rapidly and reversibly detecting histamine, preparation method of fluorescent probe and application of fluorescent probe in cell dynamic imaging

Abstract

The invention discloses a near-infrared ratio type fluorescent probe for rapidly and reversibly detecting histamine (HA), a preparation method of the fluorescent probe and application of the fluorescent probe in dynamic cell imaging, and belongs to the technical field of fluorescence imaging and biosensing. The preparation method comprises the following specific steps: synthesizing aza-BODIPY molecules (1a-1d, 2a-2d) with different halogenations, and meanwhile, connecting the aza-BODIPY molecules with hydroxyl groups at 3 and 5 sites with a hydrophilic PEG (Polyethylene Glycol) fragment and halogen to obtain an HA probe HAPCyto with good water solubility; in addition, the hydroxyl aza-BODIPY molecules are continuously subjected to substitution, halogenation and hydrolysis reaction to obtain carboxyl-containing aza-BODIPY molecules, then the aza-BODIPY molecules are subjected to amidation reaction by utilizing the site, and different organic fragments are respectively connected to obtain a cell membrane targeted HA probe HAPPM, a golgi apparatus targeted HA probe HAPGA and a HaloTag HA fluorescent probe HAPHaloTag. When HA exists in a solution, the developed probe can realize rapid reversible detection of the near-infrared ratio of histamine, also shows good reversible property in mast cells, and can realize dynamic monitoring of histamine in cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging and biosensing technology, and relates to a near-infrared ratiometric rapid reversible fluorescent probe for detecting histamine (HA), its preparation method, and its application in cell dynamic imaging. Background Technology

[0002] Neurotransmitters are chemical substances that mediate the transmission of information between neurons and play a crucial role in maintaining the physiological functions of the nervous system, such as regulating metabolism, participating in neural regulation, and influencing organ function. Histamine, as a neurotransmitter, is essential for promoting rapid immune system responses and regulating circadian rhythms. To date, designing a highly specific and rapidly reversibly detectable fluorescent probe for histamine remains a formidable challenge. First, histamine shares significant structural and chemical similarities with other neurotransmitters. Second, histamine exhibits dynamic spatiotemporal distribution changes in cells and living organisms; its production and release occur within short timeframes, but currently developed probes are generally irreversible and exhibit poor biological stability, making it difficult to dynamically track neurotransmitters. Therefore, achieving highly specific and rapidly reversibly real-time imaging of histamine in cells at the molecular level has become an extremely challenging task. In conclusion, there is an urgent need to establish a new approach to monitor real-time changes in histamine levels in cells. Summary of the Invention

[0003] To address the deficiencies and shortcomings of existing technologies, this invention innovatively provides a near-infrared ratiometric rapid reversible fluorescent probe for the detection of histamine (HA), its preparation method, and its application in dynamic cell imaging. This invention provides a novel, universal synthetic method for reversible HA fluorescent probes, synthesizing not only aza-BODIPY HA fluorescent probes with different substituents, but also obtaining HA fluorescent probes modified with different organelles (Golgi apparatus, cell membrane), cytoplasmic targeting, and HaloTag. Furthermore, the provided probes possess advantages such as reversibility, high selectivity, fast response speed, and near-infrared ratiometric response. The HA fluorescent probe described in this invention refers to a near-infrared ratiometric rapid reversible fluorescent probe for the detection of histamine (HA).

[0004] To investigate the response mechanism of the HA fluorescent probe to HA and to optimize the probe structure, this invention designed and synthesized a series of HA fluorescent probes (including but not limited to the structures shown below), the structures of which are attached. Figure 8 As shown:

[0005] Among them, the HA fluorescent probes 1a-1d and 2a-2d show that as the electronegativity of the substituents at positions 1 and 7 gradually increases, the response rate and sensitivity of the probes to HA gradually increase. This demonstrates that the detection sensitivity to HA can be adjusted by regulating the substituents at positions 1 and 7 of the HA probes.

[0006] In the HA fluorescent probes 2d and 1d, bromine atoms are introduced at positions 2 and 6 of the 2d molecule, resulting in a significant blue shift in the UV spectrum. However, due to the significant quenching effect of the bromine atoms, the fluorescence spectrum exhibits a quenching-type signal response. The HA fluorescent probe 1d molecule not only responds sensitively to HA, but also shows a significant blue shift in the UV spectrum after the reaction, and its fluorescence spectrum exhibits a ratiometric fluorescence response. Calculations revealed that the first-order reaction rate constant (k) of the HA fluorescent probe 1d molecule... obs The 1d molecule is larger than other molecules, meaning it has the fastest response rate to HA. In summary, a series of HA fluorescent probes were synthesized, and kinetic parameter testing revealed that the 1d molecule is the most sensitive to HA, and the stronger the electron-withdrawing ability at sites 1 and 7, the faster the reaction rate for HA detection.

[0007] This invention provides a near-infrared ratiometric rapid reversible fluorescent probe (HA fluorescent probe) for the detection of histamine, the structural formula of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing the HA fluorescent probe, the method comprising the following steps:

[0010] For HA fluorescent probes 1a-1d and 2a-2d, aza-BODIPY molecules modified with methoxy groups at positions 3 and 5 and different groups at positions 1 and 7 are chlorinated by reacting with N-chlorosuccinimide (NCS) at positions 2 and 6 to obtain chlorinated aza-BODIPY molecules or brominated by reacting with liquid bromine to obtain brominated aza-BODIPY molecules.

[0011] The reaction process of the preparation method is shown in the following reaction formula (a):

[0012]

[0013] Specifically, in the bromination reaction:

[0014] The bromination reaction is carried out at a temperature of 0°C to 40°C; preferably, it is carried out at 0°C.

[0015] The bromination reaction takes 5 min to 120 min; preferably, it takes 10 min.

[0016] In the bromination reaction, the molar ratio of the aza-BODIPY molecule containing methoxy groups at positions 3 and 5 and modified with different groups at positions 1 and 7 to liquid bromine is 1:(2-4); preferably, it is 1:2.

[0017] The organic solvent is selected from one or more of dichloromethane, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide (DMF), etc.; preferably, the bromination reaction is carried out using dichloromethane.

[0018] The volume of the organic solvent used in the bromination reaction is 10-100 mL; preferably, it is 45 mL.

[0019] Or, in the chlorination reaction:

[0020] The chlorination reaction is carried out at a temperature of 25°C to 80°C; preferably, at 80°C.

[0021] The chlorination reaction takes 6-12 hours; preferably, it takes 12 hours.

[0022] The chlorination reaction is preferably carried out under a nitrogen atmosphere.

[0023] In the chlorination reaction, the molar ratio of the aza-BODIPY molecule containing methoxy groups at positions 3 and 5 and modified with different groups at positions 1 and 7 to NCS is 1:(2-4); preferably, it is 1:4.

[0024] The organic solvent is selected from one or more of dichloromethane, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide (DMF), etc.; preferably, the chlorination reaction is carried out with 1,2-dichloroethane.

[0025] The volume of the organic solvent used in the chlorination reaction is 10-50 mL; preferably, it is 25 mL.

[0026] Taking the 1d molecule in reaction formula (a) as an example, while retaining the substituents at positions 1, 7 and 2, 6 of the 1d molecule, the present invention obtains HA fluorescent probe molecules with different targets and HaloTag modifications by modifying positions 3 and 5 of the 1d molecule.

[0027] This invention also provides a near-infrared ratiometric rapid reversible fluorescent probe for histamine detection that is organelle-targeting, cytoplasm-targeting, or modified with HaloTag. The structure of the fluorescent probe is as follows:

[0028]

[0029] The organelles include the Golgi apparatus and the cell membrane.

[0030] This invention also provides a method for preparing the organelle-targeting, cytoplasmic-targeting, and HaloTag-modified HA fluorescent probes, the preparation method comprising the following steps:

[0031] Aza-BODIPY molecules with F atoms at positions 1 and 7, hydroxyl groups at positions 3 and 5, and no modification at positions 2 and 6 (i.e., compound 1) were selected as the starting material. Then, a water-soluble PEG fragment was linked to positions 3 and 5 of compound 1 via a nucleophilic substitution reaction. Subsequently, chlorine atoms were modified at positions 2 and 6 of compound 5 via a chlorination reaction to obtain the cytoplasmic-targeted histamine-detecting fluorescent probe HAP. Cyto ;or,

[0032] Compound 1 was first modified by substitution, chlorination, and hydrolysis to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Then, through amidation, an organic fragment (3-(dodecylamine)propane-1-sulfonate) targeting the cell membrane was modified at positions 3 and 5 to obtain the cell membrane-targeting fluorescent probe HAP. PM ;or,

[0033] Compound 1 was first modified by substitution, chlorination, and hydrolysis to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Then, through amidation, an organic fragment (1-tetradecylamine) targeting the Golgi apparatus was modified at positions 3 and 5 to obtain the Golgi apparatus-targeting fluorescent probe HAP. GA ;or,

[0034] Compound 1 was first subjected to substitution, chlorination, and hydrolysis reactions to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Subsequently, through an amidation reaction, HaloTag chloroalkane (2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine) was modified at positions 3 and 5 to obtain the HaloTag-modified fluorescent probe HAP. HaloTag ;

[0035] The reaction process of the preparation method is shown in the following synthetic route (I):

[0036]

[0037] Specifically, in the synthetic route (I):

[0038] The specific steps for preparing compound 2 from compound 1 are as follows: in a first organic solvent, in the presence of potassium carbonate, compound 1 reacts with an ester raw material to generate compound 2.

[0039] The reaction temperature is 25℃ to 80℃; preferably, it is 80℃.

[0040] The reaction time is 6h-12h; preferably, it is 12h.

[0041] The molar ratio of compound 1, ester raw material, and potassium carbonate is 1:(2-4):(2-6); preferably, it is 1:4:2.

[0042] The ester raw material is selected from tert-butyl 2-bromoacetate.

[0043] The first organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, etc.; preferably, it is acetonitrile.

[0044] The volume of the first organic solvent is 10-50 mL; preferably, it is 50 mL.

[0045] The specific steps for preparing compound 3 from compound 2 are as follows: in a second organic solvent, compound 2 reacts with N-chlorosuccinimide to generate compound 3.

[0046] The reaction temperature is 25℃-80℃; preferably, it is 80℃.

[0047] The reaction time is 6h-12h; preferably, it is 12h.

[0048] The molar ratio of compound 2 and N-chlorosuccinimide (NCS) is 1:(2-10); preferably, it is 1:6.

[0049] The second organic solvent is selected from one or more of acetonitrile, toluene, 1,2-dichloroethane, etc.; preferably, it is 1,2-dichloroethane.

[0050] The volume of the second organic solvent is 10-50 mL; preferably, it is 20 mL.

[0051] The specific steps for preparing compound 4 from compound 3 are as follows: in a third organic solvent, compound 3 reacts with trifluoroacetic acid to generate compound 4;

[0052] The reaction temperature is 0℃ to 25℃; preferably, it is 25℃.

[0053] The reaction time is 1-6 hours; preferably, it is 3 hours.

[0054] The molar ratio of compound 3 to trifluoroacetic acid is 1:(2-10); preferably, it is 1:10.

[0055] The third organic solvent is selected from one or more of 1,2-dichloroethane, toluene, dichloromethane, etc.; preferably, it is dichloromethane.

[0056] The volume of the third organic solvent is 10-50 mL; preferably, it is 18 mL.

[0057] The specific steps for preparing compound 5 from compound 1 are as follows: in a fourth organic solvent, in the presence of potassium carbonate, compound 1 reacts with an alkoxy raw material to generate compound 5;

[0058] The reaction temperature is 25℃ to 80℃; preferably, it is 80℃.

[0059] The reaction time is 6h-12h; preferably, it is 12h.

[0060] The fourth organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, etc.; preferably, it is acetonitrile.

[0061] The molar ratio of compound 1, alkoxy raw material, and potassium carbonate is 1:(2-4):(2-6); preferably, it is 1:2:2.

[0062] The alkoxy raw material is selected from ethyl 2-(2-(2-methoxyethoxy)ethoxy-4-methylbenzenesulfonate.

[0063] The volume of the fourth organic solvent is 10-100 mL; preferably, it is 50 mL.

[0064] Among them, compound 5 was used to prepare the fluorescent probe HAP Cyto The specific steps are as follows: In the fifth organic solvent, compound 5 reacts with N-chlorosuccinimide to generate the fluorescent probe HAP. Cyto ;

[0065] The reaction temperature is 25℃-80℃; preferably, it is 80℃.

[0066] The reaction time is 6h-12h; preferably, it is 12h.

[0067] The molar ratio of compound 5 and N-chlorosuccinimide (NCS) is 1:(2-10); preferably, it is 1:6.

[0068] The fifth solvent is selected from one or more of acetonitrile, toluene, 1,2-dichloroethane, etc.; preferably, it is 1,2-dichloroethane.

[0069] The volume of the fifth organic solvent is 10-50 mL; preferably, it is 10 mL.

[0070] Among them, compound 4 was used to prepare the fluorescent probe HAP PM HAP GA HAP HaloTagThe specific steps are as follows: In the sixth organic solvent, in the presence of HATU and N,N-diisopropylethylamine, compound 4 reacts with 3-(dodecylamine)propane-1-sulfonate, 1-tetradecylamine, and 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine to generate the fluorescent probe HAP. PM HAP GA HAP HaloTag ;

[0071] The reaction temperature is 0℃~25℃; preferably, it is 25℃.

[0072] The reaction time is 6h-12h; preferably, it is 12h.

[0073] The molar ratio of compound 4, 3-(dodecylamine)propane-1-sulfonate or 1-tetradecylamine or 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine, HATU, and DIPEA is 1:(2-10):(1-4):(2-8); preferably, it is 1:5:2:6.

[0074] The sixth organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, etc.; preferably, it is N,N-dimethylformamide.

[0075] The volume of the sixth organic solvent is 5-20 mL; preferably, it is 5 mL.

[0076] In one specific embodiment of the present invention, the HA fluorescent probe preparation method provided by the present invention includes the following steps:

[0077] Compound 2: Compound 1 (0.5 g, 0.88 mmol) was dissolved in 50 mL of anhydrous acetonitrile in a 250 mL Shrek tube. Anhydrous potassium carbonate (0.24 g, 1.76 mmol) was added, followed by tert-butyl 2-bromoacetate (0.52 mL, 3.52 mmol). The mixture was refluxed overnight at 80 °C. After cooling to room temperature, the solution was extracted with dichloromethane and deionized water, dried over anhydrous Na₂SO₄, and the organic layer was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to give the green solid product, compound 2.

[0078] Compound 3: Compound 2 (0.24 g, 0.3 mmol) was added to a 100 mL Shrek tube, and 20 mL of 1,2-dichloroethane was added under a nitrogen atmosphere. (0.24 g, 1.8 mmol) N-chlorosuccinimide (NCS) was dissolved in 5 mL of 1,2-dichloroethane and injected into the tube. The mixture was stirred overnight at 80 °C. The reaction mixture was then cooled to room temperature, the solvent was removed under reduced pressure, and the crude product was extracted and purified by silica gel column chromatography to obtain the green solid product, compound 3.

[0079] Compound 4: Compound 3 (0.2 g, 0.23 mmol) was dissolved in 18 mL of dichloromethane, and trifluoroacetic acid (TFA, 1.8 mL, 23 mmol) was slowly added to the flask. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by thin-layer chromatography. After 3 h, the solvent was removed by evaporation under reduced pressure. 5 mL of dichloromethane was added, and the mixture was sonicated for 5 min. The precipitate was filtered, the residue was washed with dichloromethane, and the product was dried under reduced pressure to obtain compound 4.

[0080] Compound 5: Compound 1 (0.16 g, 0.19 mmol) and ethyl 2-(2-(2-methoxyethoxy)ethoxy-4-methylbenzenesulfonate (0.12 g, 0.38 mmol) were dissolved in 50 mL of CH3CN, and K2CO3 (0.05 g, 0.38 mmol) was added. The mixture was stirred overnight at 80 °C. The mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The product was extracted with ethyl acetate (50 mL × 3 times), the organic layer was washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum to give the crude product, which was purified by silica gel column chromatography to obtain compound 5.

[0081] HAP Cyto Compound 5 (62 mg, 0.07 mmol) was added to a 100 mL Shrek tube. 10 mL of 1,2-dichloroethane was injected into the Shrek tube under a nitrogen atmosphere, and then NCS (56 mg, 0.42 mmol) was dissolved in 5 mL of 1,2-dichloroethane. The mixture was stirred overnight at 80 °C. The mixture was cooled to room temperature, and then the solvent was removed under reduced pressure. The product was extracted with ethyl acetate (50 mL × 3 times), the organic layer was washed with saturated NaCl solution, and dried over sodium sulfate. HAP was purified by silica gel column chromatography. Cyto .

[0082] HAP PM Compound 4 (50 mg, 0.07 mmol) was dissolved in 2 mL of DMF with HATU (38 mg, 0.14 mmol) and N,N-diisopropylethylamine (DIPEA, 35 μL, 0.22 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 minutes, 3-(dodecylamine)propane-1-sulfonate (121 mg, 0.35 mmol) and 35 μL of DIPEA were dissolved in 3 mL of ultradry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After the reaction solvent was evaporated under vacuum, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. PM .

[0083] HAP GACompound 4 (20 mg, 0.03 mmol) was dissolved in 2 mL of DMF with HATU (16 mg, 0.06 mmol) and N,N-diisopropylethylamine (DIPEA, 16 μL, 0.09 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 min, 1-tetradecylamine (35 mg, 0.15 mmol) and 16 μL of DIPEA were dissolved in 3 mL of ultradry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After vacuum evaporation of the reaction solvent, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. GA .

[0084] HAP HaloTag Compound 4 (25 mg, 0.04 mmol) was dissolved in 2 mL of DMF with HATU (22 mg, 0.08 mmol) and N,N-diisopropylethylamine (DIPEA, 20 μL, 0.12 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 min, 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine (52 mg, 0.2 mmol) and 20 μL of DIPEA were dissolved in 3 mL of ultra-dry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After vacuum evaporation of the reaction solvent, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. HaloTag .

[0085] The synthesized fluorescent probe HAP Cyto HAP PM HAP GA and HAP HaloTag Both methods can achieve highly sensitive detection of HA, and the fluorescence emission peaks are all near-infrared ratiometric responses (e.g. Figure 1 As shown in the figure, HA can be quantitatively analyzed by observing the change in the ratio of the two emission peaks. Simultaneously, an HA scavenger (Ni) is added. 2+ After that, the fluorescence signal of the probe recovered (e.g. Figure 1 As shown in the figure, the probe can achieve reversible detection of HA. Compared with other probes, the HA near-infrared fluorescence probe of the present invention has good selectivity, good biocompatibility, fast response speed, high stability, and quantitative analysis capability, making the detection more accurate. The reversible detection property gives the probe great advantages in biosensing, dynamic fluorescence imaging and other fields.

[0086] The present invention also provides the application of the fluorescent probe in the detection of HA in vitro and / or intracellular and / or in vivo; the detection of HA is achieved by single-photon fluorescence detection; wherein the cells include, but are not limited to, mast cells; and the in vivo does not include humans.

[0087] The present invention also provides the application of the fluorescent probe in biosensing and dynamic fluorescence imaging.

[0088] Because the fluorescent probe can react with HA to alter the molecular conjugated structure of aza-BODIPY, it leads to a change in the position of the probe's fluorescence emission peak. This invention also provides a method for the in vitro reversible fluorescence detection of histamine, the method comprising the following steps: reacting the fluorescent probe as described above with histamine in a buffer mixture, then exciting the solution with an excitation wavelength of 400-450 nm, and measuring the fluorescence intensity of the solution. Simultaneously, a histamine scavenger, Ni, is added. 2+ The fluorescence intensity change of the fluorescent probe is then measured. Preferably, the excitation wavelength is 405 nm.

[0089] Specifically, with the increase of histamine content, the fluorescent probes all exhibit ratiometric changes with good linearity, thereby achieving quantitative detection of histamine. This is further demonstrated by the addition of Ni. 2+ Subsequently, the fluorescence of the fluorescent probe recovered, proving that the fluorescent probe can achieve reversible detection of histamine.

[0090] The buffer solution is a phosphate buffer, preferably with a concentration of 0.2 mM and a pH of 7.4.

[0091] The reaction temperature is 0-25℃; preferably, it is 25℃.

[0092] The reaction time is 1s-5s; preferably, it is 1.8s.

[0093] The linear range of the method is 0 μM-275 μM; the limit of detection is 89 nM.

[0094] The linear relationship between the single-photon fluorescence intensity ratio and the HA concentration is R. 2 = 0.899-0.996; preferably, R 2 =0.996.

[0095] The method can also be applied to cells and living organisms.

[0096] Specifically, using the fluorescent probe HAP of the present invention Cyto For example, the method includes the following steps: in a buffer solution, the fluorescent probe HAP of the present invention is added... Cyto Different concentrations of HA were gradually added to the solution, and the changes in fluorescence intensity of the probe and the new substance were measured at an excitation wavelength of 405 nm, thereby achieving quantitative detection of HA.

[0097] In one specific embodiment of the present invention, the in vitro fluorescence detection method for HA specifically includes: taking the fluorescent probe prepared by the above method, diluting it to 10 μM with phosphate buffer, adding different equivalents of HA solution each time, and then measuring the fluorescence intensity of the solution at an excitation wavelength of 405 nm; as the concentration of HA increases, the fluorescence emission peak of the fluorescent probe shows a blue shift, and the HA can be quantitatively analyzed by the change in the ratio of the two emission peaks (e.g., ...). Figure 2 As shown), and exhibits good linearity within the linear range. Furthermore, the fluorescent probe enables rapid and reversible detection of HA (e.g., Figure 1 As shown in the figure, this is beneficial for the probe to achieve dynamic monitoring of histamine under physiological conditions.

[0098] Taking HA fluorescent probe 1d as an example, the products after the reaction were characterized by NMR and mass spectrometry. For the series of probes developed in this invention, a new emission peak appeared after reaction with HA, while the original fluorescence emission peak decreased, indicating that the product maintained a considerable degree of conjugation. Figure 3 As shown, firstly, combined with the nuclear magnetic resonance spectrum (NMR spectrum) Figure 3 AB), HA fluorescent probe 1d was dissolved in deuterated dimethyl sulfoxide solvent. The 1H NMR spectrum of HA fluorescent probe 1d after the addition of 2 equivalents of HA showed significant splitting of the H atoms in the aromatic region, indicating the formation of an asymmetric addition product. Furthermore, the 1C NMR spectrum of the product revealed a new high-field peak, confirming that the reaction between HA fluorescent probe 1d and HA is a nucleophilic addition reaction. High-resolution mass spectrometry (HR-MS) (…) Figure 3 C) It was also clarified that the HA fluorescent probe 1d forms a 1:1 addition product with HA.

[0099] In one specific embodiment of the present invention, a cytoplasmic-targeted HA fluorescent probe HAP is used. Cyto For example, through in vitro experiments, the interference of neurotransmitters, thiol compounds, and amino acids on the detection was investigated. Figure 4 It is known that common neurotransmitters, amino acids, and thiol compounds have no significant effect on HA detection (interference <10%). The selectivity and anti-interference experiments show that the HA fluorescent probe in this invention has good selectivity and anti-interference ability.

[0100] In one specific embodiment of the present invention, in order to examine the targeting ability of the developed fluorescent probe, cultured mast cells were subjected to different targeting HA fluorescent probes HAP. Cyto HAP PM and HAP GAIncubate for 15 min, then add commercial probes and incubate for another 15 min. Aspirate the cell culture medium, add PBS buffer, and use a confocal microscope to determine cell morphology. Excite the cells with a 405 nm laser, simultaneously opening the green and red channels for fluorescence imaging (e.g., ...). Figure 5 As shown in the image, cell fluorescence images were processed using ImageJ software to obtain HA fluorescent probes HAP targeting different targets. PM HAP Cyto and HAP GA The colocalization coefficients with commercial probes were 0.94, 0.86, and 0.84, respectively, which demonstrates that the developed targeting probes have excellent targeting performance.

[0101] This invention also provides a method for dynamic fluorescence imaging of intracellular histamine, comprising the following steps: co-incubating cells with the fluorescent probe described above, then adding a buffer solution, observing using a confocal microscope, exciting the cells with an excitation wavelength of 400-450 nm, and observing the real-time dynamic fluorescence imaging and intensity values ​​of the cells after exogenous histamine stimulation, thereby realizing the fluorescence response to intracellular histamine and performing dynamic fluorescence imaging analysis. Preferably, the excitation wavelength is 405 nm; preferably, the concentration of the phosphate buffer solution is 0.2 mM and the pH is 7.4.

[0102] In one specific embodiment of the present invention, the dynamic fluorescence imaging method for intracellular HA uses a cytoplasmic HA fluorescent probe HAP. Cyto For example, intracellular experiments include the following steps: exposing P815 mast cells to the cytoplasm-targeting HA fluorescent probe HAP. Cyto The cells were co-incubated, then buffer was added, and observation was performed using a fluorescence confocal microscope. Excitation was conducted at a wavelength of 405 nm. After exogenous addition of HA, real-time dynamic observation was performed to observe the fluorescence emission at 720 nm. As HA decreased, the fluorescence emission of the fluorescent probe at approximately 580 nm gradually increased, yielding a ratiometric response. This allowed for ratiometric fluorescence response to intracellular HA and imaging analysis. Simultaneously, a HA scavenger (Ni) was added after HA incubation. 2+ The observed recovery of fluorescence demonstrates that the probe can reversibly detect HA, which strongly suggests that the fluorescent probe can achieve dynamic and reversible monitoring of intracellular HA (e.g., Figure 6 (As shown).

[0103] This invention also provides a fluorescent probe HAP HaloTag In cell imaging, the application includes the following steps: pre-transfecting P815 mast cells with a Golgi targeting protein containing a tag, and incubating the cells with the fluorescent probe HAP. HaloTagSimultaneously, the fluorescent probe HAP HaloTag The fluorescent probe HAP was incubated in cells transfected with a protein lacking Golgi targeting and simultaneously subjected to confocal fluorescence microscopy imaging. HaloTag It achieves targeting of organelles and has better stability compared to organic probes. Under the same conditions, it exhibits slower fluorescence decay in living cells compared to small organic molecules.

[0104] In one specific embodiment of the present invention, in order to examine the developed fluorescent probe HAP HaloTag In bioimaging applications, P815 mast cells were pre-transfected with Golgi targeting protein (ST) containing the tag, and the cells were incubated with the fluorescent probe HAP. HaloTag Simultaneously, the fluorescent probe HAP HaloTag The developed fluorescent probe HAP was incubated in cells transfected with a protein lacking Golgi apparatus targeting and simultaneously subjected to confocal microscopy fluorescence imaging. HaloTag The probe can target organelles and has better stability compared to organic probes, exhibiting high fluorescence intensity for 3 days (e.g., Figure 7 As shown in the figure, it has a wider range of applications in the detection of HA.

[0105] The incubation temperature is 25-37℃, preferably 37℃; the incubation time between the cells and the HA probe is 10min-60min, preferably 30min.

[0106] The present invention also provides the application of the fluorescent probes described above, or the preparation methods described above, the methods described above, or the dynamic fluorescence imaging methods described above in the in vitro and / or intracellular detection of histamine, cell imaging and biosensing.

[0107] The beneficial effects of this invention include: firstly, the synthesis of a series of reversible near-infrared fluorescent probes with high selectivity for HA, wherein the HA fluorescent probes exhibit significant fluorescence ratio changes under 405nm excitation, which can correct for environmental interference, improve the accuracy of HA quantitative analysis, and enrich detection methods. To broaden the application of probes in biology and improve their water solubility and targeting ability, HA fluorescent probes with different organelles (Golgi apparatus, cell membrane), cytoplasmic targeting, and HaloTag modification were subsequently synthesized by modifying the 3 and 5 sites of the aza-BODIPY molecule. These molecules all possess good targeting ability (…). Figure 5 Furthermore, the reversible HA fluorescent probe developed can realize dynamic monitoring of HA in cells, indicating that the fluorescent probe described in this invention has a good application prospect. Attached Figure Description

[0108] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0109] Figure 1 It is a fluorescent probe HAP Cyto HAP PM HAP GA and HAP HaloTag Changes in fluorescence spectra after gradual addition of HA and fluorescence kinetics of four probes: (A) Fluorescent probe HAP Cyto (B) Fluorescence spectrum changes after adding HA to phosphate buffer; PM Changes in fluorescence spectrum after adding HA to phosphate buffer; (C) Fluorescent probe HAP GA Changes in fluorescence spectrum after adding HA to phosphate buffer; (D) Fluorescent probe HAP HaloTag Changes in fluorescence spectrum after adding HA to phosphate buffer; (E) Fluorescent probe HAP Cyto Add HA and Ni to phosphate buffer 2+ Post-fluorescence spectroscopy kinetics test; (F) fluorescent probe HAP PM Add HA and Ni to phosphate buffer 2+ Post-fluorescence spectroscopy kinetics; (G) fluorescent probe HAP GA Add HA and Ni to phosphate buffer 2+ Post-fluorescence spectroscopy kinetics assay; (H) fluorescent probe HAP HaloTag Add HA and Ni to phosphate buffer 2+ Post-fluorescence spectroscopy kinetics test; wherein, the excitation wavelength for the above spectral tests is 405 nm.

[0110] Figure 2 It is a fluorescent probe HAP Cyto、 HAP PM HAP GA HAP HaloTag Linear relationship of fluorescence ratio changes for different concentrations of HA.

[0111] Figure 3The following are characterization images of the product after the reaction of HA fluorescent probe 1d with HA: (A) Proton NMR spectrum of the product after the reaction of HA fluorescent probe 1d with HA, wherein the solvent is deuterated dimethyl sulfoxide and HA is 2 equivalents; (B) Carbon NMR spectrum of the product after the reaction of HA fluorescent probe 1d with HA, wherein the solvent is deuterated dimethyl sulfoxide and the newly generated high-field peak is marked with an asterisk; (C) High-resolution mass spectrum of the product after the reaction of HA fluorescent probe 1d with HA.

[0112] Figure 4 HAP is a cytoplasmic-targeted HA fluorescent probe. Cyto Selectivity radar chart and interference experiment for HA detection: (A) represents the selective experiment when interfering neurotransmitters, thiol compounds, and amino acids are present alone; (B) represents the interference experiment when HA coexists with potential interfering neurotransmitters, thiol compounds, and amino acids; the bar chart represents the interference experiment; the excitation wavelength is 405 nm.

[0113] Figure 5 These are different targeted HA fluorescent probes HAP Cyto HAP PM HAP GA Fluorescence images of co-localization with different commercially available targeting probes in P815 mast cells; the excitation wavelength was 405 nm.

[0114] Figure 6 HAP is a cytoplasmic-targeted HA fluorescent probe. Cyto After incubating P815 mast cells, exogenous HA and Ni were added. 2+ Dynamic fluorescence imaging after stimulation (A) and real-time intensity map (B), where the excitation wavelength is 405 nm and the scale bar is 10 μm.

[0115] Figure 7 It is a HaloTag-modified HA fluorescent probe HAP HaloTag The real-time fluorescence intensity bar graph after incubating live P815 cells expressing Golgi protein ST is shown in Figure B. Cells transfected with empty vector are used as a negative control in Figure A. The excitation wavelength is 405 nm. Figure 8 This is a schematic diagram of the structure of the HA fluorescent probe. Detailed Implementation

[0116] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0117] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0118] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0119] This invention provides a method for preparing a near-infrared ratiometric rapid reversible histamine (HA) detection fluorescent probe and its application in live-cell dynamic imaging, belonging to the field of fluorescence imaging and biosensing technology. This invention also discloses the preparation methods of different targeted HA probes and Halo Tag HA probes. The specific steps are as follows: First, referring to the conventional synthesis method of aza-BODIPY, aza-BODIPY molecules with hydroxyl groups at positions 3 and 5 are synthesized. Then, a hydrophilic PEG fragment and a halogen are linked through substitution and halogenation reactions to obtain the HA probe HAP with good water solubility. Cyto Furthermore, the obtained hydroxyl aza-BODIPY molecules were further subjected to substitution, halogenation, and hydrolysis reactions to obtain carboxyl-containing aza-BODIPY molecules. These carboxyl-containing molecules were then subjected to amidation at this site to connect with a cell membrane-targeting organic fragment, resulting in the cell membrane-targeting HA probe HAP. PM By linking organic fragments that target the Golgi apparatus, a Golgi apparatus-targeted HA probe, HAP, was obtained. GA HaloTag chloroalkanes were linked to obtain the HaloTag HA fluorescent probe HAP. HaloTag When HA is present in solution, the developed probes can achieve rapid and reversible near-infrared ratio detection of histamine (HA), and also exhibit good reversible properties in P815 mast cells, enabling dynamic monitoring of histamine in cells.

[0120] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents.

[0121] Example 1. Preparation of HA fluorescent probes 2a-2d and 1a-1d

[0122] For HA fluorescent probes 1a-1d and 2a-2d, aza-BODIPY molecules modified with methoxy groups at positions 3 and 5 and different groups at positions 1 and 7 were reacted with N-chlorosuccinimide (NCS) and liquid bromine at positions 2 and 6, respectively, to obtain chlorinated and brominated aza-BODIPY molecules. The synthesis method for 1a-1d was the same. Aza-BODIPY molecules (0.07 mmol) modified with methoxy groups at positions 3 and 5 and different groups at positions 1 and 7 were added to a 100 mL Shrek tube and dissolved in 20 mL of 1,2-dichloroethane under a nitrogen atmosphere. NCS (37 mg, 0.28 mmol) was dissolved in 5 mL of 1,2-dichloroethane and injected into the reaction system. The mixture was stirred overnight at 80 °C. Afterwards, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The precipitate was redissolved in dichloromethane and washed with deionized water (3 × 100 mL) and brine (1 × 100 mL). The organic layer was dried on anhydrous sodium sulfate, concentrated, and then separated by column chromatography to obtain the product. The synthesis of molecules 2a-2d was similar; 0.1 mmol of aza-BODIPY molecules modified with methoxy groups at positions 3 and 5, but with different groups at positions 1 and 7, was dissolved in 40 mL of dichloromethane at 0 °C. Liquid bromine solution (12 μL, 0.2 mmol) was then added dropwise to 5 mL of dichloromethane. The reaction was monitored by thin-layer chromatography, and quenched with sodium thiosulfate solution (2 M, 10 mL). The mixture was washed with deionized water (3 × 100 mL) and brine (1 × 100 mL), and then extracted with dichloromethane. The organic layer was dried on anhydrous sodium sulfate, concentrated, and then separated by column chromatography to obtain the product.

[0123] The proton and carbon spectra of the HA fluorescent probes 2a-2d and 1a-1d prepared in this embodiment are as follows:

[0124] HA fluorescent probe 2a, 1 HNMR (600MHz, CDCl3, ppm) δ7.86 (dd, J=7.3, 2.1Hz, 4H), 7.77 (d,

[0125] J=8.7Hz,4H),7.50-7.37(m,6H),6.99(d,J=8.8Hz,4H),3.86(s,6H). 13 C NMR (126MHz, CDCl3, ppm) δ161.76,157.54,144.20,142.54,132.49,130.79,130.72,129.49,128.00,121.80,113.62,110.23,55.34.HR-MS(ESI)m / z for C 34 H24 BBr2F2N3NaO2[M+Na] + :736.0189.Found:736.0201.

[0126] HA fluorescent probe 2b, 1 H NMR (600MHz, CDCl3, ppm) δ7.78 (d, J=8.8Hz, 4H), 7.74 (d, J=

[0127] 8.5Hz,4H),7.61(d,J=8.5Hz,4H),7.01(d,J=8.8Hz,4H),3.89(s,6H). 13 C NMR (126MHz, CDCl3, ppm) δ162.01,161.23,140.47,139.18,135.33,132.58,132.11,131.38,129.54,124.29,121.45,113.68,55.13.HR-MS(ESI)m / z for C 34 H 22 BBr4F2N3NaO2[M+Na] + :772.0000.Found:772.0014.

[0128] HA fluorescent probe 2c, 1 H NMR (600MHz, CDCl3, ppm) δ7.81 (d, J=8.5Hz, 4H), 7.78 (d, J=

[0129] 8.8Hz,4H),7.45(d,J=8.5Hz,4H),7.02(d,J=12.7Hz,4H),3.89(s,6H). 13 C NMR (126MHz, CDCl3, ppm) δ161.92,157.78,144.11,141.22,135.89,132.50,131.91,129.11,128.41,127.05,121.58,113.67,55.35.HR-MS(ESI)m / z for C 34 H 22 BBr2Cl2F2N3NaO2[M+Na] + :805.9496.Found:805.9428.

[0130] HA fluorescent probe 2d, 1 H NMR (600MHz, CDCl3, ppm) δ7.92–7.80 (m, 4H), 7.76 (d, J = 8.8

[0131] Hz, 4H), 7.14 (t, J = 8.6 Hz, 4H), 6.99 (d, J = 8.8 Hz, 4H), 3.86 (s, 6H). 13 C NMR (126 MHz, CDCl3, ppm) δ 162.80, 161.86, 142.95, 135.91, 132.73, 132.68, 132.49, 124.62, 121.65, 115.32, 115.18, 113.66, 55.35. HR-MS (ESI) m / z for C 34 H 22 BBr2F4N3NaO2 [M+Na] + : 891.8507. Found: 891.8529.

[0132] HA fluorescent probe 1a, 1 H NMR (600 MHz, CDCl3, ppm) δ 7.90 (d, J = 6.6 Hz, 4H), 7.82 (d, J =

[0133] 8.7 Hz, 4H), 7.52–7.38 (m, 6H), 7.00 (d, J = 8.8 Hz, 4H), 3.86 (s, 6H). 13 C NMR (126 MHz, CDCl3, ppm) δ 161.87, 156.06, 143.40, 139.41, 132.52, 130.73, 130.12, 129.49, 128.12, 121.85, 121.02, 113.77, 55.44. HR-MS (ESI) m / z for C 34 H 24 BCl2F2N3NaO2 [M+Na] + : 648.1199. Found: 648.1212.

[0134] HA fluorescent probe 1b, 1 H NMR (600 MHz, CDCl3, ppm) δ 7.83 (d, J = 8.8 Hz, 4H), 7.79 (d, J =

[0135] 8.5 Hz, 4H), 7.62 (d, J = 8.5 Hz, 4H), 7.02 (d, J = 8.9 Hz, 4H), 3.89 (s, 6H). 1313C NMR (126 MHz, CDCl3, ppm) δ 162.00, 161.88, 157.43, 138.03, 132.58, 132.06, 131.48, 129.03, 125.82, 120.72, 113.82, 110.79, 55.36. HR-MS (ESI) m / z for C 34 H 22 BBr2Cl2F2N3NaO2 [M + Na] + : 805.9496. Found: 805.9402.

[0136] HA fluorescent probe 1c, 1 1H NMR (600 MHz, CDCl3, ppm) δ 7.86 (d, J = 8.5 Hz, 4H), 7.83 (d, J =

[0137] 8.5 Hz, 4H), 7.46 (d, J = 8.3 Hz, 4H), 7.02 (d, J = 8.5 Hz, 4H), 3.89 (s, 6H). 13 13C NMR (126 MHz, CDCl3, ppm) δ 162.04, 135.90, 132.55, 131.84, 130.34, 130.09, 128.99, 128.52, 128.43, 114.58, 113.82, 113.67, 55.38. HR-MS (ESI) m / z for C 34 H 22 BCl4F2N3NaO2 [M + Na] + : 716.0527. Found: 716.0526.

[0138] HA fluorescent probe 1d, 1 1H NMR (600 MHz, CDCl3, ppm) δ 7.88 (dd, J = 8.6, 5.5 Hz, 4H), 7.81 (d,

[0139] J = 8.7 Hz, 4H), 7.15 (d, J = 8.6 Hz, 4H), 7.00 (d, J = 8.8 Hz, 4H), 3.86 (s, 6H). 13 13C NMR (126 MHz, CDCl3, ppm) δ 162.67, 161.97, 156.15, 143.26, 138.28, 132.62, 132.51, 126.18, 120.87, 115.44, 115.30, 113.81, 55.37. HR-MS (ESI) m / z for C 34 H 22BCl2F4N3NaO2[M+Na] + :684.1010.Found:684.1017.

[0140] Example 2. Fluorescent probe HAP Cyto HAP PM HAP GA and HAP HaloTag

[0141] The aza-BODIPY molecule (compound 1) with hydroxyl groups at positions 3 and 5 was selected for use as a cytoplasmic-targeting HA fluorescent probe, HAP. Cyto The synthesis of the HA probe involved linking a water-soluble PEG fragment at positions 3 and 5 via nucleophilic substitution, followed by chlorination to modify chlorine atoms at positions 2 and 6. For the synthesis of cell membrane and Golgi apparatus HA probes, aza-BODIPY molecules (compound 4) with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5 were first obtained through substitution, chlorination, and hydrolysis. Organic fragments targeting cell membranes and the Golgi apparatus were then modified at positions 3 and 5 to obtain the cell membrane-targeted HA fluorescent probe HAP. PM And the Golgi-targeted HA fluorescent probe HAP GA Modifying HaloTag chloroalkane at positions 3 and 5 yields the HaloTag-modified HA fluorescent probe HAP. HaloTag .

[0142] The HA fluorescent probe preparation method provided by the present invention includes the following steps:

[0143] Compound 2: Compound 1 (0.5 g, 0.88 mmol) was dissolved in 50 mL of anhydrous acetonitrile in a 250 mL Shrek tube. Anhydrous potassium carbonate (0.24 g, 1.76 mmol) was added, followed by tert-butyl 2-bromoacetate (0.52 mL, 3.52 mmol). The mixture was refluxed overnight at 80 °C. After cooling to room temperature, the solution was extracted with dichloromethane and deionized water, dried over anhydrous Na₂SO₄, and the organic layer was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to give the green solid product, compound 2.

[0144] Compound 3: Compound 2 (0.24 g, 0.3 mmol) was added to a 100 mL Shrek tube, and 20 mL of 1,2-dichloroethane was added under a nitrogen atmosphere. (0.24 g, 1.8 mmol) N-chlorosuccinimide (NCS) was dissolved in 5 mL of 1,2-dichloroethane and injected into the tube. The mixture was stirred overnight at 80 °C. The reaction mixture was then cooled to room temperature, the solvent was removed under reduced pressure, and the crude product was extracted and purified by silica gel column chromatography to obtain the green solid product, compound 3.

[0145] Compound 4: Compound 3 (0.2 g, 0.23 mmol) was dissolved in 18 mL of dichloromethane, and trifluoroacetic acid (TFA, 1.8 mL, 23 mmol) was slowly added to the flask. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by thin-layer chromatography. After 3 h, the solvent was removed by evaporation under reduced pressure. 5 mL of dichloromethane was added, and the mixture was sonicated for 5 min. The precipitate was filtered, the residue was washed with dichloromethane, and the product was dried under reduced pressure to obtain compound 4.

[0146] Compound 5: Compound 1 (0.16 g, 0.19 mmol) and ethyl 2-(2-(2-methoxyethoxy)ethoxy-4-methylbenzenesulfonate (0.12 g, 0.38 mmol) were dissolved in 50 mL of CH3CN, and K2CO3 (0.05 g, 0.38 mmol) was added. The mixture was stirred overnight at 80 °C. The mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The product was extracted with ethyl acetate (50 mL × 3 times), the organic layer was washed with saturated NaCl solution, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum to give the crude product, which was purified by silica gel column chromatography to obtain compound 5.

[0147] HAP Cyto Compound 5 (62 mg, 0.07 mmol) was added to a 100 mL Shrek tube. 10 mL of 1,2-dichloroethane was injected into the Shrek tube under a nitrogen atmosphere, and then NCS (56 mg, 0.42 mmol) was dissolved in 5 mL of 1,2-dichloroethane. The mixture was stirred overnight at 80 °C. The mixture was cooled to room temperature, and then the solvent was removed under reduced pressure. The product was extracted with ethyl acetate (50 mL × 3 times), the organic layer was washed with saturated NaCl solution, and dried over sodium sulfate. HAP was purified by silica gel column chromatography. Cyto .

[0148] HAP PM Compound 4 (50 mg, 0.07 mmol) was dissolved in 2 mL of DMF with HATU (38 mg, 0.14 mmol) and N,N-diisopropylethylamine (DIPEA, 35 μL, 0.22 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 minutes, 3-(dodecylamine)propane-1-sulfonate (121 mg, 0.35 mmol) and 35 μL of DIPEA were dissolved in 3 mL of ultradry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After the reaction solvent was evaporated under vacuum, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. PM .

[0149] HAP GACompound 4 (20 mg, 0.03 mmol) was dissolved in 2 mL of DMF with HATU (16 mg, 0.06 mmol) and N,N-diisopropylethylamine (DIPEA, 16 μL, 0.09 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 min, 1-tetradecylamine (35 mg, 0.15 mmol) and 16 μL of DIPEA were dissolved in 3 mL of ultradry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After vacuum evaporation of the reaction solvent, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. GA .

[0150] HAP HaloTag Compound 4 (25 mg, 0.04 mmol) was dissolved in 2 mL of DMF with HATU (22 mg, 0.08 mmol) and N,N-diisopropylethylamine (DIPEA, 20 μL, 0.12 mmol) under a nitrogen atmosphere. After stirring at room temperature for 5 min, 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine (52 mg, 0.2 mmol) and 20 μL of DIPEA were dissolved in 3 mL of ultra-dry DMF and injected into the system. The mixture was stirred at room temperature for 12 h. After the reaction solvent was evaporated under vacuum, the product was purified by silica gel column chromatography to obtain the green product, namely the fluorescent probe HAP. HaloTag .

[0151] In this invention, the proton and carbon spectral data of the compounds prepared by the above method are as follows:

[0152] Compound 2: 1 H NMR (600MHz, CDCl3, ppm) δ8.06 (d, J=8.9Hz, 4H), 8.03-7.99 (m, 4H), 7.15 (t, J= 8.6,12Hz,4H),7.00(s,2H),6.99(d,J=3.4Hz,4H),4.59(s,4H),1.51(s,18H). 13 CNMR (151MHz, CDCl3, ppm) δ167.41,164.28,162.62,160.20,158.05,145.11,142.05,131 .56,130.99,128.49,124.68,118.35,115.66,114.77,82.66,65.56,27.99.HRMS(ESI):C 44 H 40 BF4N3O6[M+Na] + calcd 816.2844,found:816.2858.

[0153] Compound 3: 11H NMR (600 MHz, CDCl3, ppm) δ 7.92 - 7.88 (m, 4H), 7.82 (d, J = 8.8 Hz, 4H), 7.20 - 7.15 (m, 4H), 7.00 (d, J = 8.9 Hz, 4H), 4.57 (s, 4H), 1.49 (s, 18H). 13 13C NMR (151 MHz, CDCl3, ppm) δ 177.24, 171.17, 167.49, 164.36, 162.69, 160.26, 156.04, 143.28, 138.39, 132.68, 132.63, 132.50, 126.15, 121.66, 115.46, 115.31, 114.37, 82.76, 65.67, 28.04. HRMS (ESI): C 44 H 38 BCl2F4N3O6 [M + Na] + calcd 884.2167, found: 884.2107.

[0154] Compound 4: 1 1H NMR (600 MHz, DMSO-d6, ppm) δ 7.95 (dd, J = 8.4, 5.6 Hz, 4H), 7.74 (d, J = 8.7 Hz, 4H), 7.46 (t, J = 8.8 Hz, 4H), 7.10 (d, J = 8.8 Hz, 4H), 4.82 (s, 4H). 13 13C NMR (151 MHz, DMSO-d6, ppm) δ 170.28, 164.30, 162.64, 160.71, 156.05, 143.22, 138.46, 133.36, 133.31, 133.12, 132.71, 126.17, 121.89, 121.07, 116.18, 116.04, 114.84, 114.40, 64.98, 28.16. HRMS (ESI): C 36 H 22 BCl2F4N3O6 [M + Na] + calcd 772.0813, found: 772.0828.

[0155] Compound 5: 1H NMR(600MHz,CDCl3,ppm)δ8.05(d,J=8.8Hz,8H),7.04–6.96(m,8H),6.93(s,2H),4.25–4.20(m,4H),3.94–3.90(m,4H),3.78(dd,J=5.7,3.7Hz,4H),3.69(dd,J=5.7,3.7Hz,4H),3.66(dd,J=5.6,3.6Hz,4H),3.57(dd,J=5.7,3.6Hz,4H),3.40(s,6H). 13 CNMR(126MHz,CDCl3,ppm)δ160.91,160.11,157.63,145.19,142.70,131.47,130.76,125.31,124.51,117.02,114.72,114.59,71.95,70.88,70.67,70.60,69.60,67.51,63.61,59.06,14.84.HRMS(ESI):C 48 H 52 BF4N4O8[M+CH3CN+H] + calcd 899.3814,found:899.4177.

[0156] HAP Cyto : 1 H NMR(600MHz,CDCl3,ppm)δ7.96(d,J=8.8Hz,4H),7.80(d,J=8.6Hz,

[0157] 4H),7.02(dd,J=8.7,1.6Hz,8H),4.24–4.19(m,4H),3.91–3.88(m,4H),3.79–3.76(m,4H),3.74–3.70(m,4H),3.70–3.66(m,4H),3.60–3.56(m,4H),3.41(s,6H). 13 C NMR(126MHz,CDCl3,ppm)δ160.90,160.80,160.24,155.64,143.23,138.95,132.34,131.36,123.02,121.34,114.21,113.75,71.96,70.89,70.69,70.61,69.58,67.46,63.63,59.06,55.42,14.81.HRMS(ESI):C 46 H 46 BCl2F4N3O8[M+H]+ calcd 926.2769,found:926.3667.

[0158] HAP PM : 1 H NMR(600MHz,MeOD,ppm)δ7.91(dd,J=8.8,5.4Hz,4H),7.79(d,J=8.7

[0159] Hz,4H),7.22(t,J=8.8,12Hz,4H),7.11-7.05(m,4H),4.88(s,4H),3.71(dp,J=13.2,6.6Hz,4H),3.58-3.51(m,8H),3.44-3.36(m,8H),3.23-3.19(m,4H),3.17(t,J=7.2,12Hz,4H),3.00-2.96(m,4H),2.89-2.85(m,4H),2.84-2.78(m,4H),2.05(dd,J=29.9,10.1Hz,4H),1.58(m,4H),0.91-0.87(m,8H),0.87–0.83(m,6H). 13 C NMR(151MHz,MeOD,ppm)δ166.95,163.38,161.75,160.97,146.86,136.75,132.87,130.37,130.11,127.42,115.95,115.57,115.42,49.35,47.07,47.00,31.77,29.59,29.51,29.41,29.21,29.18,22.57,14.38.HRMS(ESI):C 66 H 82 BCl2F4N5O 10 S2 2- [M] 2- calcd 662.7460,found:662.7168.

[0160] HAP GA : 1 H NMR(600MHz,CDCl3,ppm)δ7.91(dd,J=8.3,5.6Hz,4H),7.85(d,J=8.6

[0161] Hz,4H),7.18(t,J=8.5Hz,4H),7.05(d,J=8.7Hz,4H),6.56(s,2H),4.56(s,4H),1.36–1.25(m,52H),0.90(t,J=6.9Hz,6H). 13 C NMR(151MHz,CDCl3,ppm)δ167.33,159.31,155.91,143.35,138.68,132.69,132.64,126.01,122.26,115.54,115.39,114.47,67.28,39.19,32.00,29.71,29.70,29.67,29.61,29.56,29.38,29.34,29.27,26.88,22.71,14.14.HRMS(ESI):C 64 H 80 BCl2F4N5O4[M+Na] + calcd 1162.5514,found:1162.5546.

[0162] HAP HaloTag : 1 H NMR(600MHz,CDCl3,ppm)δ7.90(dd,J=8.3,5.6Hz,4H),7.83(d,J=

[0163] 8.6Hz,4H),7.18(t,J=8.5Hz,4H),7.05(d,J=8.5Hz,4H),4.57(s,4H),3.60(dd,J=11.1,7.1Hz,12H),3.57(d,J=4.7Hz,4H),3.52(t,J=6.7Hz,4H),3.47(t,J=6.6Hz,4H),1.80–1.73(m,4H),1.65–1.58(m,4H),1.45(dt,J=13.9,7.1Hz,4H),1.40–1.34(m,4H). 13 C NMR(151MHz,CDCl3,ppm)δ167.52,162.77,159.35,155.94,143.34,138.65,132.64,126.03,122.21,115.53,115.39,114.47,71.30,70.43,70.06,69.71,67.26,45.05,38.90,32.51,29.71,29.46,26.68,25.42.HRMS(ESI):C 56 H 62BCl4F4N5O8[M+Na] + calcd1184.3250,found:1184.3265.

[0164] Example 3. Detection of HA in vitro

[0165] In vitro sample detection and preparation of fluorescence intensity calibration curves

[0166] The fluorescent probe HAP prepared in Example 2 of this invention was used. Cyto HAP PM HAP GA HAP HaloTag Dilute the solution to 10 μM with phosphate buffer. Add 2 mL of the solution to different equivalents of HA solution each time, and then measure the fluorescence intensity of the solution at an excitation wavelength of 405 nm. As the concentration of HA increases, the fluorescence intensity of the fluorescent probe HAP increases. Cyto HAP PM HAP GA HAP HaloTag As the HA concentration increases, the fluorescence emission peaks of the probe all exhibit a ratiometric response. The change in the ratio of the two emission peaks allows for quantitative analysis of HA. Figure 2 It exhibits good linearity and low detection limit, making it suitable for effective detection of HA.

[0167] Example 4. The cytoplasmic-targeted HA fluorescent probe HAP prepared in Example 2 of the present invention Cyto Selectivity and anti-interference ability

[0168] To evaluate the cytoplasmic-targeted HA fluorescent probe HAP prepared in Example 2 of this invention Cyto The selectivity and anti-interference ability of fluorescence signals were investigated by examining the changes in fluorescence signals of common neurotransmitters (HA, NE, DA, Gly, E, 5-HT, GABA, Glu, Asp, Ach), thiol compounds (Cys, Hcy, GSH), and amino acids (Lys, Tyr, his, Thr, Ala, Met, Arg) both individually and in co-existence with HA. Figure 4 It is known that common neurotransmitters, amino acids, and thiol compounds have no significant effect on HA detection (interference <10%). The selectivity and anti-interference experiments show that the HA fluorescent probe in this invention has good selectivity and anti-interference ability.

[0169] Example 5. Co-localization experiment of HA fluorescent probes with different targets and dynamic fluorescence imaging of intracellular HA

[0170] Different HA fluorescent probes HAP prepared in Example 2 of this invention Cyto HAP PMHAP GA Colocalization experiments with commercial probes in mast cells and confocal dynamic fluorescence imaging of HA ( Figure 6 ).

[0171] For the colocalization experiment, cultured mast cells were incubated with the described fluorescent probe for 15 min, followed by incubation with the commercial probe for another 15 min. The cell culture medium was then aspirated, and PBS buffer was added. Cell morphology was then assessed using a confocal microscope, and cells were excited with fluorescence at an excitation wavelength of 405 nm. Simultaneously, the green and red channels were opened for fluorescence imaging. The cell fluorescence images were processed using ImageJ software, yielding colocalization coefficients of 0.94, 0.86, and 0.84 for the HA fluorescent probe and the commercial probe, respectively, indicating that the invented targeting probe possesses excellent targeting ability.

[0172] For the cell dynamic fluorescence imaging experiment, cultured mast cells were incubated with the fluorescent probe for 15 min, then the cell culture medium was aspirated, PBS buffer was added, cell morphology was assessed using a confocal microscope, and cells were excited with fluorescence at an excitation wavelength of 405 nm. With the addition of 100 μM HA, the fluorescence at 720 nm of the probe gradually decreased, while the fluorescence at 580 nm gradually increased. With the addition of 100 μM HA scavenger (Ni... 2+ With the addition of ), the fluorescence at 720nm of the probe in the cell gradually increased, while the fluorescence at 580nm gradually decreased, and fluorescence recovery occurred.

[0173] Example 6. Preparation of HaloTag-modified HA fluorescent probe HAP in Example 2 of the present invention HaloTag confocal fluorescence imaging

[0174] P815 mast cells were pre-transfected with Golgi targeting protein (ST) containing the tag, while cells transfected with the empty vector served as a negative control. The fluorescent probe HAP was then used. HaloTag The developed fluorescent probe HAP was incubated separately into transfected live cells, and simultaneously subjected to confocal microscopy fluorescence imaging. HaloTag It can target the Golgi apparatus and has better stability than organic probes. The blank group showed almost complete fluorescence quenching after one day due to the instability of organic molecules, while the experimental group showed very little fluorescence decay over three days, still suitable for bioimaging. Figure 7 Therefore, the HaloTag-modified HA fluorescent probe HAP used in this invention for detecting HA... HaloTag It has broader application prospects.

[0175] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0176] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0177] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0178] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A near-infrared ratiometric rapid reversible fluorescent probe for the detection of histamine, characterized in that, The structural formula of the fluorescent probe is shown below:

2. A near-infrared ratiometric rapid reversible fluorescent probe for histamine detection, characterized by targeting organelles, targeting cytoplasm, or being modified with HaloTag, wherein... The structure of the fluorescent probe is as follows:

3. A method for preparing a fluorescent probe as described in claim 1, characterized in that, The preparation method includes the following steps: chloro-aza-BODIPY molecules containing methoxy groups at positions 3 and 5 and modified with different groups at positions 1 and 7 are reacted with N-chlorosuccinimide at positions 2 and 6 to obtain chloro-aza-BODIPY molecules, or brominated with liquid bromine to obtain brominated aza-BODIPY molecules. The reaction process of the preparation method is shown in the following reaction formula (a):

4. The preparation method according to claim 3, characterized in that, In the bromination reaction The bromination reaction is carried out at a temperature of 0°C to 40°C; and / or, the bromination reaction takes 5 min to 120 min; and / or, the molar ratio of the aza-BODIPY molecule containing methoxy groups at positions 3 and 5 and modified with different groups at positions 1 and 7 to liquid bromine in the bromination reaction is 1:(2-4); and / or, the organic solvent for the bromination reaction is selected from one or more of dichloromethane, 1,2-dichloroethane, acetonitrile, and N,N-dimethylformamide; and / or, the volume of the organic solvent for the bromination reaction is 10-100 mL. Or, in the chlorination reaction, The chlorination reaction is carried out at a temperature of 25°C-80°C; and / or, the chlorination reaction takes 6-12 hours; and / or, the molar ratio of the aza-BODIPY molecule modified with methoxy groups at positions 3 and 5 and different groups at positions 1 and 7 to N-chlorosuccinimide is 1:(2-4); and / or, the organic solvent for the chlorination reaction is selected from one or more of dichloromethane, 1,2-dichloroethane, acetonitrile, and N,N-dimethylformamide; and / or, the volume of the organic solvent for the chlorination reaction is 10-50 mL.

5. A method for preparing a fluorescent probe as described in claim 2, characterized in that, The preparation method includes the following steps: Aza-BODIPY molecules with F atoms at positions 1 and 7, hydroxyl groups at positions 3 and 5, and no modification at positions 2 and 6 (i.e., compound 1) were selected as the starting material. Then, a water-soluble PEG fragment was linked to positions 3 and 5 of compound 1 via a nucleophilic substitution reaction. Subsequently, chlorine atoms were modified at positions 2 and 6 of compound 5 via a chlorination reaction to obtain the cytoplasmic-targeted histamine-detecting fluorescent probe HAP. Cyto ;or, Compound 1 was first subjected to substitution, chlorination, and hydrolysis reactions to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Subsequently, through amidation, an organic fragment targeting the cell membrane was modified at positions 3 and 5 to obtain the cell membrane-targeting fluorescent probe HAP. PM ;or, Compound 1 was first modified by substitution, chlorination, and hydrolysis to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Then, through amidation, an organic fragment targeting the Golgi apparatus was modified at positions 3 and 5 to obtain the Golgi apparatus-targeting fluorescent probe HAP. GA ;or, Compound 1 was first subjected to substitution, chlorination, and hydrolysis reactions to obtain an aza-BODIPY molecule, namely compound 4, with chlorine atoms modified at positions 2 and 6 and carboxyl groups modified at positions 3 and 5. Subsequently, through an amidation reaction, HaloTag chloroalkane was modified at positions 3 and 5 to obtain the HaloTag-modified fluorescent probe HAP. HaloTag ; The reaction process of the preparation method is shown in the following synthetic route (I):

6. The method as described in claim 5, characterized in that, In the synthetic route (I), The specific steps for preparing compound 2 from compound 1 are as follows: in a first organic solvent, in the presence of potassium carbonate, compound 1 reacts with an ester raw material to generate compound 2; Wherein, the reaction temperature is 25℃~80℃; and / or, the reaction time is 6h-12h; and / or, the molar ratio of compound 1, ester raw material, and potassium carbonate is 1:(2-4):(2-6); and / or, the first organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, and N,N-dimethylformamide; and / or, the volume of the first organic solvent is 10-50mL; and / or, the ester raw material is selected from tert-butyl 2-bromoacetate; And / or, The specific steps for preparing compound 3 from compound 2 are as follows: in a second organic solvent, compound 2 reacts with N-chlorosuccinimide to generate compound 3; Wherein, the reaction temperature is 25℃-80℃; and / or, the reaction time is 6h-12h; and / or, the molar ratio of compound 2 to N-chlorosuccinimide is 1:(2-10); and / or, the second organic solvent is selected from one or more of acetonitrile, toluene, and 1,2-dichloroethane; and / or, the volume of the second organic solvent is 10-50mL; And / or, The specific steps for preparing compound 4 from compound 3 are as follows: in a third organic solvent, compound 3 reacts with trifluoroacetic acid to generate compound 4; Wherein, the reaction temperature is 0℃~25℃; and / or, the reaction time is 1h-6h; and / or, the molar ratio of compound 3 to trifluoroacetic acid is 1:(2-10); and / or, the third organic solvent is selected from one or more of 1,2-dichloroethane, toluene, and dichloromethane; and / or, the volume of the third organic solvent is 10-50mL; And / or, The specific steps for preparing compound 5 from compound 1 are as follows: in a fourth organic solvent, in the presence of potassium carbonate, compound 1 reacts with an alkoxy-based raw material to generate compound 5; Wherein, the reaction temperature is 25℃~80℃; and / or, the reaction time is 6h-12h; and / or, the fourth organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, and N,N-dimethylformamide; and / or, the molar ratio of compound 1, alkoxy raw material, and potassium carbonate is 1:(2-4):(2-6); and / or, the volume of the fourth organic solvent is 10-100mL; And / or, The fluorescent probe HAP was prepared using compound 5. Cyto The specific steps are as follows: In the fifth organic solvent, compound 5 reacts with N-chlorosuccinimide to generate the fluorescent probe HAP. Cyto ; Wherein, the reaction temperature is 25℃-80℃; and / or, the reaction time is 6h-12h; and / or, the molar ratio of compound 5 to N-chlorosuccinimide is 1:(2-10); and / or, the fifth organic solvent is selected from one or more of acetonitrile, toluene, and 1,2-dichloroethane; and / or, the volume of the fifth organic solvent is 10-50mL; And / or, The fluorescent probe HAP was prepared using compound 4. PM HAP GA HAP HaloTag The specific steps are as follows: In the sixth organic solvent, in the presence of HATU and N,N-diisopropylethylamine, compound 4 reacts with 3-(dodecylamine)propane-1-sulfonate, 1-tetradecylamine, and 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine to generate the fluorescent probe HAP. PM HAP GA HAP HaloTag ; Wherein, the reaction temperature is 0℃~25℃; and / or, the sixth organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, and N,N-dimethylformamide; and / or, the reaction time is 6h-12h; and / or, the molar ratio of compound 4, 3-(dodecylamine)propane-1-sulfonate or 1-tetradecylamine or 2-(2-(6-chlorohexyl)oxyethoxy)ethoxy-1-amine, HATU, and DIPEA is 1:(2-10):(1-4):(2-8); and / or, the volume of the sixth organic solvent is 5-20mL.

7. The method described below, characterized in that, The method includes: (1) A method for in vitro fluorescent reversible detection of histamine, the method comprising the following steps: reacting the fluorescent probe as described in claim 2 with histamine in a buffer solution, then exciting the solution with an excitation wavelength of 400-450 nm, and measuring the fluorescence intensity of the solution. Simultaneously, a histamine scavenger, Ni, is added. 2+ Continue to measure the change in fluorescence intensity of the fluorescent probe; (2) A method for dynamic fluorescence imaging of intracellular histamine, the method comprising the following steps: incubating cells with the fluorescent probe as described in claim 2, then adding buffer solution, observing using a confocal microscope, exciting with an excitation wavelength of 400-450 nm, observing real-time dynamic fluorescence imaging and intensity values ​​of cells after stimulation by exogenous histamine, thereby realizing the fluorescence response to intracellular histamine and performing dynamic fluorescence imaging analysis.

8. The method as described in claim 7, characterized in that, In method (1), as the content of added histamine increases, the fluorescent probes all exhibit ratiometric changes with good linearity, thereby achieving quantitative detection of histamine. When Ni is added... 2+ Subsequently, the fluorescence of the fluorescent probe recovered, proving that the fluorescent probe can achieve reversible detection of histamine.

9. A fluorescent probe HAP HaloTag Its application in cell imaging is characterized by, The application includes the following steps: pre-transfecting P815 mast cells with a Golgi targeting protein containing Tag, and incubating the cells with the fluorescent probe HAP as described in claim 2. HaloTag Simultaneously, the fluorescent probe HAP HaloTag The fluorescent probe HAP was incubated in cells transfected with a protein lacking Golgi targeting and simultaneously subjected to confocal fluorescence microscopy imaging. HaloTag It achieves targeting of organelles and has better stability compared to organic probes. Under the same conditions, it exhibits slower fluorescence decay in living cells compared to small organic molecules.

10. The application of the fluorescent probe as described in claim 1, or the fluorescent probe as described in claim 2, or the preparation method as described in claim 3 or 4, or the preparation method as described in claim 5 or 6, or the method as described in claim 7 or 8 in in vitro and / or intracellular detection of histamine, cell imaging and biosensing, cell dynamic imaging, cell transfection, and Halo Tag cell imaging technology.

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