A hemoglobin fluorescent probe, preparation method and application thereof

Abstract

The present invention relates to a hemoglobin fluorescent probe, a preparation method and its use. The chemical formula of the probe is: (C 37 H 28 The fluorescent probe, described in this paper, provides a low-background fluorescence signal when bound to hemoglobin (N3OI). Through specific metabolic processes or competitive displacement between the probe and nonsteroidal anti-inflammatory drugs (NSAIDs), the probe can be effectively dissociated from hemoglobin. This makes it suitable for controllable hemoglobin-mediated bioimaging, particularly for monitoring the distribution and dynamic changes of hemoglobin within organisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a hemoglobin fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hemoglobin is the main protein in red blood cells. Abnormal hemoglobin concentration is associated with a variety of diseases, such as anemia, liver disease, kidney disease, and malignant tumors. In-depth research on its structure and function is crucial for understanding life processes and disease mechanisms. Therefore, a deep understanding of the structure and function of hemoglobin is extremely important for revealing the mysteries of life processes and the mechanisms of disease. Bovine hemoglobin and human hemoglobin are extremely similar in structure and spectral properties. Because bovine hemoglobin is easier to obtain, it is often used as a hemoglobin model for various spectroscopic studies and mechanism analyses. At present, although there are many methods for the detection of hemoglobin, such as electrophoresis, chromatography, and colorimetry, most of these methods can only be performed in a laboratory environment and often cannot achieve real-time dynamic monitoring of hemoglobin concentration.

[0003] Fluorescent probes, with their unique optical properties, such as high sensitivity, high selectivity, rapid response, and visual imaging, provide new possibilities for real-time monitoring of hemoglobin. By designing and synthesizing specific fluorescent probes for hemoglobin, real-time, in situ, and non-invasive monitoring of hemoglobin concentration can be achieved, providing more accurate and timely information for early diagnosis and treatment of diseases. Although there have been some reports on the research of hemoglobin fluorescent probes, there is still significant room for improvement in the sensitivity and selectivity of existing probes. Especially in complex physiological environments, such as when patients take drugs with strong plasma protein binding ability, these probes face challenges in maintaining their stability and accuracy. Therefore, it is of great significance to develop new hemoglobin fluorescent probes with higher sensitivity and selectivity to promote the development of hemoglobin monitoring technology. Summary of the Invention

[0004] In response to the problems existing in the prior art for hemoglobin detection, the present invention provides a hemoglobin fluorescent probe with high sensitivity, high signal-to-noise ratio, and controllable fluorescence signal, which can realize in vitro hemoglobin detection or hemoglobin-mediated precise and controllable fluorescence imaging in cells.

[0005] To achieve the above objectives, the present invention first provides a hemoglobin fluorescent probe, characterized in that it has the following structural formula:

[0006]

[0007] The fluorescent probe of the present invention can provide a low-background fluorescence signal when bound to hemoglobin. Through specific metabolic processes or competitive substitution between the probe and nonsteroidal anti-inflammatory drugs, the probe can be effectively dissociated from hemoglobin. It is suitable for hemoglobin-mediated controllable biological imaging and has broad application prospects, especially in monitoring the distribution and dynamic changes of hemoglobin in organisms. Compared with existing technologies, the hemoglobin fluorescent probe of the present invention has the following advantages:

[0008] (1) The hemoglobin fluorescent probe provided by the present invention has strong near-infrared fluorescence. In the presence of multiple proteins including hemoglobin, the probe fluorescence is significantly quenched only by hemoglobin, showing strong specificity.

[0009] (2) It has high response sensitivity, strong protein binding ability and anti-interference ability;

[0010] (3) The probe binds to hemoglobin through a non-covalent interaction. Through a specific metabolic process or competitive substitution between the non-steroidal anti-inflammatory drug ibuprofen and the probe, the probe can be effectively dissociated from hemoglobin, achieving a controllable fluorescence response of the probe to hemoglobin.

[0011] To further achieve the above-mentioned object of the present invention, the present invention also provides a method for preparing the above-mentioned hemoglobin fluorescent probe:

[0012] The steps include:

[0013] Step 1: Mix 4-pyridineboronic acid, tetrakis(triphenylphosphine)palladium, and K2CO3 with 1,4-dioxane and H2O, then add 4'-(4-bromophenyl)acetophenone, stir, and heat under reflux under nitrogen protection; after cooling, separate by column chromatography to obtain 4-(4-pyridyl)-biphenyl-4'-ethanone; the reaction formula is as follows:

[0014]

[0015] Step 2: Dissolve the 4-(4-pyridyl)-biphenyl-4'-ethanone and malononitrile prepared in step 1 in toluene, then add ammonium acetate and acetic acid, stir and heat under reflux, cool, and separate by column chromatography to obtain 2-isocyano-3-[4-(4-pyridyl)biphenyl-4'-yl]but-2-enenitrile; the reaction formula is as follows:

[0016]

[0017] Step 3: Potassium tert-butoxide and (formylmethylene)triphenylphosphine were stirred in a toluene solvent on an ice bath, added to 6-methoxy-2-naphthaldehyde, heated under reflux under nitrogen protection, cooled, and separated by column chromatography to obtain 3-(6-methoxynaphthalen-2-yl)propenal; the reaction formula is as follows:

[0018]

[0019] Step 4: Dissolve the 2-isocyano-3-[4-(4-pyridinyl)biphenyl-4'-yl]but-2-enenitrile prepared in step 3, 3-(6-methoxynaphthalen-2-yl)acrolein prepared in step 3, and piperidine in dichloromethane, stir and heat under reflux, cool, and separate by column chromatography to obtain 2-isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile; the reaction formula is as follows:

[0020]

[0021] Step 5: Dissolve 2-isocyanato-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile and iodomethane prepared in acetonitrile, stir and heat under reflux, cool and filter, and elute with anhydrous ether to obtain the hemoglobin fluorescent probe; the reaction formula is as follows:

[0022]

[0023] Furthermore, the specific process of step 1 is:

[0024] 1.1 Mix 4-pyridineboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 with 1,4-dioxane and H2O, and stir to form a mixed solution;

[0025] 1.2 Dissolve 4′-(4-bromophenyl)acetophenone in 1,4-dioxane and mix with the mixture from step 1.1. Pour nitrogen into the mixture and stir at 80°C for 8 hours. Monitor the reaction progress by TLC until the starting material 4′-(4-bromophenyl)acetophenone is completely consumed.

[0026] 1.3 After the reaction is completed, the mixture is cooled to room temperature, and the reactant is extracted three times with water and dichloromethane. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product is purified on a silica gel column to obtain 4-(4-pyridyl)-biphenyl-4'-ethanone;

[0027] The molar ratio of 4-pyridineboric acid, tetrakis(triphenylphosphine)palladium, K2CO3 and 4′-(4-bromophenyl)acetophenone is 3:0.05:6:2; the volume ratio of 1,4-dioxane and H2O is 4:1, wherein the molar volume ratio of tetrakis(triphenylphosphine)palladium to 1,4-dioxane is 3.1-3.2 mmol / L.

[0028] The specific synthesis process of step 2 is:

[0029] 4-(4-pyridyl)-biphenyl-4'-ethanone and malononitrile were dissolved in toluene, and then ammonium acetate and acetic acid were added and refluxed for 12 hours. After the reaction was completed as monitored by TLC, the mixture was cooled to room temperature and extracted with water and dichloromethane. After concentration under reduced pressure, the crude product was purified on a silica gel column to obtain 2-isocyano-3-[4-(4-pyridyl)biphenyl-4'-yl]but-2-enenitrile. The molar ratio of 4-(4-pyridyl)-biphenyl-4'-ethanone, malononitrile, ammonium acetate, and acetic acid was 14:15.4:4.62:13.02.

[0030] The specific synthesis process of step 3 is:

[0031] Potassium tert-butoxide and (formylmethylene)triphenylphosphine are stirred in toluene in an ice bath for 1 to 2 hours, then slowly added dropwise to 6-methoxy-2-naphthaldehyde, mixed, and reacted at 50°C for 24 hours under nitrogen protection. After the reaction, the mixture is quenched with 30% dilute hydrochloric acid, and then extracted with water and ethyl acetate. After evaporation of the solvent, the crude product is purified on a silica gel column to obtain 3-(6-methoxynaphth-2-yl)propenal; the molar ratio of 6-methoxy-2-naphthaldehyde, potassium tert-butoxide, and (formylmethylene)triphenylphosphine is 3:6:3.3.

[0032] The specific synthesis process of step 4 is as follows: 2-isocyano-3-[4-(4-pyridinyl)biphenyl-4'-yl]but-2-enenitrile and 3-(6-methoxynaphthalen-2-yl)acrolein in a molar ratio of 0.9:1 are mixed, dissolved in dichloromethane with piperidine, and refluxed for 6 to 8 hours. After concentration under reduced pressure, the crude product is purified on a silica gel column to obtain 2-isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile.

[0033] The synthesis process of step 5 is as follows: 2-isocyanato-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile and iodomethane 1 in a molar ratio of 0.43:1.3 are dissolved in acetonitrile and refluxed for 24 hours. After concentration under reduced pressure, acetonitrile is added until dissolved, precipitated with ether, filtered and washed to obtain the target hemoglobin fluorescent probe.

[0034] Furthermore, in the aforementioned steps, the silica gel column is a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:2.

[0035] The present invention also provides an application of the hemoglobin fluorescent probe in hemoglobin detection or hemoglobin-mediated intracellular controllable fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is the nuclear magnetic hydrogen spectrum of the fluorescent probe of the present invention, and the solvent is d6-DMSO.

[0038] Figure 2 This is the nuclear magnetic carbon spectrum of the fluorescent probe of the present invention, and the solvent is d6-DMSO.

[0039] Figure 3 The comparison of fluorescence intensity changes of fluorescent probes in different proteins (λ ex =520nm).

[0040] Figure 4 The comparison of fluorescence intensity changes of fluorescent probe in different concentrations of bovine hemoglobin (λ ex =520nm).

[0041] Figure 5 is the fluorescence quenching diagram of the fluorescent probe to bovine hemoglobin (λ ex =280nm).

[0042] Figure 6 This paper studies the effects of various common commercial drugs on the responsiveness of the fluorescent probe bovine hemoglobin.

[0043] Figure 7 The effect of different concentrations of ibuprofen on the response ability of the fluorescent probe bovine hemoglobin.

[0044] Figure 8 This is a diagram of the HepG2 cell toxicity test of the fluorescent probe and the complex of the fluorescent probe and bovine hemoglobin.

[0045] Figure 9 Laser confocal fluorescence imaging of HepG2 cells stained with fluorescent probe, complex of fluorescent probe and bovine hemoglobin, and complex of fluorescent probe and bovine hemoglobin with ibuprofen added after 4h, 12h, and 24h respectively (λ ex =543nm). DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation methods of the present invention are further described below.

[0047] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0048] The present invention provides a protein structure-dependent hemoglobin fluorescent probe, the structural formula of which is:

[0049]

[0050] The synthetic route of the hemoglobin probe of the above structure is as follows:

[0051]

[0052] The present invention is further described below by way of examples.

[0053] Example 1 Preparation of hemoglobin fluorescent probe:

[0054] Step 1: Add 3 mmol of 0.37 g of 4-pyridineboronic acid, 0.05 mmol of 0.06 g of tetrakis(triphenylphosphine)palladium, and 6 mmol of 0.83 g of K2CO3 to a 50 mL Schlenk tube. Then, add 16 mL of 1,4-dioxane and 4 mL of H2O. Dissolve 2 mmol of 0.55 g of 4′-(4-bromophenyl)acetophenone in 1,4-dioxane and add the mixture to the Schlenk tube. Under nitrogen, stir the mixture at 80°C for 8 hours, and monitor the reaction progress by TLC. After the reaction is complete, cool to room temperature and extract three times with water and dichloromethane. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product is purified on a silica gel column (ethyl acetate / dichloromethane, 1 / 2, v / v) to yield 4-(4-pyridyl)-biphenyl-4'-ethanone; its H NMR spectrum parameters are: 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.64 (s, 2H), 8.00 (d, J = 8.4Hz, 2H), 7.70 (s, 4H), 7.67 (d, J = 8.4Hz, 2H), 7.53 (d, J = 6.0Hz, 2H), 2.59 (s, 3H).

[0055] Step 2: Dissolve 0.39g 4-(4-pyridyl)-biphenyl-4'-ethanone 1.4mmol and 0.10g malononitrile 1.54mmol in 20mL toluene. Then add 0.35g ammonium acetate 4.62mmol and 0.74mL acetic acid 13.02mmol, and reflux for 12 hours. Monitor the reaction progress by TLC. After the reaction is completed, cool to room temperature and extract with water and dichloromethane. After concentration under reduced pressure, the crude product is purified on a silica gel column (ethyl acetate / petroleum ether, 2 / 1, v / v) to obtain 2-isocyano-3-[4-(4-pyridyl)biphenyl-4'-yl]but-2-enenitrile; its H NMR spectrum parameters are: 1 H NMR (400MHz, CDCl3) δ (ppm): 8.67 (s, 2H), 8.00 (d, J = 8.4Hz, 2H), 7.70 (s, 4H), 7 .68(d,J=2.0Hz,1H),7.66(t,J=1.6Hz,1H),7.53(d,J=6.0Hz,2H),2.59(s,3H).

[0056] Step 3: Add 0.69 g of 6-methoxy-2-naphthaldehyde 3 mmol to a Schlenk tube. Stir 0.66 g of potassium tert-butoxide 6 mmol and 0.99 g of (formylmethylene)triphenylphosphine 3.3 mmol in toluene in an ice bath for one to two hours and then slowly add dropwise to the Schlenk tube using a syringe. Protect with nitrogen and react at 50 ° C for 24 hours. After the reaction is completed, quench with 10 mL of 30% dilute hydrochloric acid. Then extract with water and ethyl acetate. After evaporation of the solvent, the crude product is purified on a silica gel column (ethyl acetate / petroleum ether, 1 / 1, v / v) to obtain 3-(6-methoxynaphthalene-2-yl)propenal; its nuclear magnetic hydrogen spectrum parameters are: 1 H NMR (400MHz, CDCl3) δ (ppm): 9.73 (d, J = 7.6Hz, 1H), 7.91 (s, 1H), 7.77 (t, J = 9.2 Hz,2H),7.66-7.58(m,2H),7.21-7.15(m,2H),6.82-6.76(m,1H),3.95(s,3H).

[0057] Step 4: Dissolve 0.29 g of 2-isocyano-3-[4-(4-pyridinyl)biphenyl-4'-yl]but-2-enenitrile (0.9 mmol), 0.23 g of 3-(6-methoxynaphthalen-2-yl)acrolein (0.99 mmol), and 15 drops of piperidine in 20 mL of dichloromethane and reflux for 6 h. After concentration under reduced pressure, the crude product was purified on a silica gel column (ethyl acetate / petroleum ether, 2 / 1, v / v) to obtain 2-isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile; its H NMR spectrum parameters are:1 H NMR (400MHz, CDCl3) δ (ppm): 8.67 (d, J = 5.2Hz, 2H), 8.11 (d, J = 8.0Hz, 2H), 7.92 (t, J = 11.6Hz, 3H) ,7.72(d,J=8.8Hz,7H),7.55(t,J=12.0Hz,4H),7.18(s,2H),7.10(d,J=8.8Hz,2H),3.88(s,3H).

[0058] 0.22 g of 2-isocyanato-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile (0.43 mmol) and 0.18 g of iodomethane (1.3 mmol) were dissolved in 20 mL of acetonitrile and refluxed for 24 h. After concentration under reduced pressure, a small amount of acetonitrile was added to dissolve the mixture, precipitated with ether, filtered, and washed to obtain the target probe. Its H NMR spectrum parameters are: 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 9.04 (d, J = 6.8 Hz, 2H), 8.61 (d, J = 6.8 Hz, 2H), 8.28 (t, J = 14.4 Hz, 2H), 8.18-8.07 (m, 4H), 7.96-7.73 (m, 4H), 7.71-7.46 (m, 3H), 7.40-7.26 (m, 2H), 7.22-7.01 (m, 2H), 6.96-6.87 (m, 1H), 4.36 (s, 3H), 3.90-3.79 (m, 3H). Its C NMR parameters are: 13 C NMR(101MHz,DMSO-d6)δ(ppm):170.4,170.3,159.8,153.3,149.9,146.3,143.6,137.0,136.7,136.6,136.2,132.6,131 .1,130.8,129.9,129.1,129.0,128.6,128.4,125.0,124.7,124.6,123.4,120.1,114.5,113.6,106.9,81.4,56.0,47.8.

[0059] Figure 1 This is the H NMR spectrum of the hemoglobin probe synthesized in the above embodiment of the present invention, the solvent is d6-DMSO; Figure 2 This is the nuclear magnetic carbon spectrum of the fluorescent probe of the present invention, and the solvent is d6-DMSO.

[0060] Example 2 Fluorescent probe protein selective response test:

[0061] Stock solution preparation: The probe was dissolved in DMSO, and hemin was dissolved in acidic acetone to prepare a stock solution with a concentration of 2 mM. Pepsin was dissolved in 0.1 M phosphate buffered saline (PBS), pH 2.0. Bovine hemoglobin, trypsin, lysozyme, α-chymotrypsin, and γ-globulin were dissolved in 0.1 M PBS, pH 7.4 to prepare a stock solution with a concentration of 80 μM.

[0062] In a 5 ml volumetric flask, bovine hemoglobin, hemin, pepsin, trypsin, lysozyme, α-chymotrypsin, and γ-globulin were first added to a final concentration of 5 μM. The probe was then added to a final concentration of 5 μM, and the mixed solution was brought to volume using 0.1 M PBS buffer. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectrum of the sample was measured using an F-7000 fluorescence spectrophotometer with an excitation wavelength of 520 nm and excitation and emission slits set to 5 nm and 10 nm, respectively.

[0063] like Figure 3 As shown, the probe itself has strong intrinsic fluorescence. After adding bovine hemoglobin to the probe, the probe fluorescence was significantly quenched. However, under the same conditions, the probe was added with pepsin, trypsin, lysozyme, α-chymotrypsin, γ-globulin Example 3 Fluorescent probe hemoglobin response sensitivity test.

[0064] In a 5 ml volumetric flask, bovine hemoglobin was first added to a final concentration of 0-7 μM, followed by the probe at a final concentration of 5 μM. The mixed solution was then brought to volume using 0.1 M PBS, pH 7.4. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectrum of the sample was measured using an F-7000 fluorescence spectrophotometer with an excitation wavelength of 520 nm and excitation and emission slits set to 5 nm and 10 nm, respectively.

[0065] like Figure 4 As shown in Figure 2, the intrinsic fluorescence of the probe is significantly quenched as the concentration of bovine hemoglobin increases. When the molar ratio of bovine hemoglobin to probe concentration reaches 1:1, the intrinsic fluorescence of the probe no longer changes. Figure 4 As can be seen from the inset, the change in fluorescence intensity (ΔF) of the probe at an emission wavelength of 640 nm shows a good linear correlation (R 2 =0.9988). Based on the signal-to-noise ratio of S / N=3, the detection limit of the probe for bovine hemoglobin was calculated to be 0.26 μM.

[0066] Example 4 Fluorescence quenching test of bovine hemoglobin by fluorescent probe

[0067] In a 5 ml volumetric flask, bovine hemoglobin was first added to a final concentration of 5 μM, followed by the probe at a final concentration of 0-14 μM. The mixed solution was then brought to volume using 0.1 M PBS, pH 7.4. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectrum of the sample was measured using an F-7000 fluorescence spectrophotometer with an excitation wavelength of 280 nm and excitation and emission slits set to 5 nm and 5 nm, respectively.

[0068] like Figure 5 As shown in Figure 2, the intrinsic fluorescence of bovine hemoglobin was significantly quenched as the probe concentration increased. Figure 5 As can be seen from the inset, the fluorescence intensity of bovine hemoglobin at an emission wavelength of 330 nm showed a good linear correlation with the probe concentration (0-14 μM) within a certain range (R 2 =0.9934). According to the classic Stern-Volmer equation, the association constant between the probe and bovine hemoglobin was calculated to be 6.96×10 4 M -1 .

[0069] Example 5: Test on the Effect of Common Commercial Drugs on the Response Ability of Fluorescent Probe Bovine Hemoglobin

[0070] Preparation of common commercial drug stock solutions: Ibuprofen, phenylbutazone, ibrutinib, anastrozole, erlotinib, regorafenib, nandrolone, capecitabine, palbociclib, zaltoprofen, lidocaine, moxifloxacin hydrochloride, posaconazole, aripiprazole, repaglinide, trelagliptin, epalrestat, simvastatin, azilsartan, and telaprevir are dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1 mM.

[0071] In a 5 ml volumetric flask, bovine hemoglobin and a probe at a final concentration of 5 μM were first added, followed by the addition of various commercial drugs (ibuprofen, phenylbutazone, ibrutinib, anastrozole, erlotinib, regorafenib, nandrolone, capecitabine, palbociclib, zaltoprofen, lidocaine, moxifloxacin hydrochloride, posaconazole, aripiprazole, repaglinide, trelagliptin, epalrestat, simvastatin, azilsartan, and telaprevir) at a final concentration of 5 μM. The mixed solution was then brought to volume using 0.1 M PBS, pH 7.4. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectra of the samples were measured using an F-7000 fluorescence spectrophotometer with an excitation wavelength of 520 nm and excitation and emission slits set to 5 nm and 10 nm, respectively.

[0072] like Figure 6As shown, except for the classic non-steroidal anti-inflammatory drug ibuprofen, which affects the fluorescence response performance of the probe to bovine hemoglobin, various other commercial drugs (phenylbutazone, ibrutinib, anastrozole, erlotinib, regorafenib, nandrolone, capecitabine, palbociclib, zaltoprofen, lidocaine, moxifloxacin hydrochloride, posaconazole, aripiprazole, repaglinide, trelagliptin, epalrestat, simvastatin, azilsartan, and telaprevir) have no significant effect on the fluorescence response performance of the probe to bovine hemoglobin, indicating that the probe has strong anti-interference ability and is suitable for accurate and reliable fluorescence response of the probe to hemoglobin in the context of patients receiving multiple types of drug treatment.

[0073] Example 6: Effect of Ibuprofen on the Response Ability of Fluorescent Probe Bovine Hemoglobin

[0074] In a 5 ml volumetric flask, bovine hemoglobin and the probe were first added to a final concentration of 5 μM. Subsequently, ibuprofen was added to a final concentration of 0-10 μM, and the mixed solution was brought to volume using 0.1 M PBS, pH 7.4. The solution was allowed to stand for at least 30 minutes. The fluorescence emission spectrum of the sample was measured using an F-7000 fluorescence spectrophotometer with an excitation wavelength of 520 nm and excitation and emission slits set to 5 nm and 10 nm, respectively.

[0075] like Figure 7 As shown, with the increase of ibuprofen concentration, the quenched fluorescence of the probe after binding to bovine hemoglobin gradually recovered, indicating that ibuprofen can be used to regulate the fluorescence response of the probe to hemoglobin.

[0076] Example 7 Cytotoxicity Test

[0077] The seeding density in 6-well plates was 1×10 4 Different concentrations of probes and complexes of probes and bovine hemoglobin (molar ratio 1:1) were added to the corresponding wells and incubated in a 37°C, 5% CO2 incubator for 24 hours. The cytotoxicity of the samples was determined using the MTT assay.

[0078] like Figure 8 As shown, as the concentration of the probe and the complex of the probe and bovine hemoglobin (molar ratio 1:1) increased, the liver cancer cell HepG2 maintained extremely high cell activity, indicating that the probe had very low cytotoxicity.

[0079] Example 8 Live Cell Imaging Assay

[0080] HepG2 liver cancer cells were inoculated into laser confocal culture dishes and cultured overnight in a cell culture incubator (37°C, 5% CO2). Three groups were set up in the experiment: the first group only added the probe with a final concentration of 5 μM; the second group added a complex of the probe and bovine hemoglobin (molar ratio 1:1) with a final concentration of 5 μM; the third group, on the basis of adding a complex of the probe and bovine hemoglobin (molar ratio 1:1) with a final concentration of 5 μM, additionally added ibuprofen with a final concentration of 5 μM after culturing for 1 hour. Subsequently, each group of cells was cultured under the same cell culture conditions, starting from the time point of probe addition, for 4 hours, 12 hours and 24 hours respectively. The culture medium was removed and the cells were washed three times with PBS buffer. The fluorescence imaging effect of the treated HepG2 cells was observed using a laser confocal microscope (excitation wavelength 543 nm).

[0081] like Figure 9 As shown in Figure 2, the probe exhibited good cell imaging performance in HepG2 cells. However, after incubation of HepG2 cells with a complex of the probe and bovine hemoglobin (1:1 molar ratio) for 4 hours, no fluorescence signal was observed within the cells. With increasing incubation time, a significant fluorescence signal from the probe was gradually detected within the cells, a phenomenon attributed to the recovery of the probe fluorescence signal during hemoglobin metabolism.

[0082] At the time of probe addition, the changes in intracellular fluorescence signal were compared before and after the addition of ibuprofen to a complex of the probe and bovine hemoglobin (molar ratio 1:1). The experiment observed that the presence of ibuprofen significantly enhanced the intracellular fluorescence signal. Combined with an in-depth analysis of the effect of ibuprofen on the responsiveness of the fluorescent probe to bovine hemoglobin, it was confirmed that ibuprofen can effectively dissociate the probe from hemoglobin, thereby achieving precise regulation of the probe's hemoglobin fluorescence properties.

[0083] In summary, the hemoglobin fluorescent probe of the present invention, its preparation method, and use are synthesized based on simple chemical raw materials, have selective hemoglobin quenching response, hemoglobin concentration-dependent response characteristics, and can effectively resist the interference of various common commercial drugs on its response performance. Ibuprofen can be used to achieve precise regulation of the hemoglobin fluorescence properties of the probe. Therefore, the fluorescent probe of the present invention is suitable for accurate detection of hemoglobin in vitro and accurate and controllable fluorescence imaging of hemoglobin-mediated cells for non-therapeutic or diagnostic purposes.

[0084] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and exemplary embodiments. They can be applied to a variety of fields suitable for the present invention. Further modifications will be readily apparent to those skilled in the art. Therefore, the present invention is not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims

1. A hemoglobin fluorescent probe, characterized in that: It has the following structural formula: 。 2. A method for preparing the hemoglobin fluorescent probe according to claim 1, characterized in that: The steps include: Step 1: Mix 4-pyridineboronic acid, tetrakis(triphenylphosphine)palladium, and K2CO3 with 1,4-dioxane and H2O, then add 4'-(4-bromophenyl)acetophenone, stir, and heat under reflux under nitrogen protection; after cooling, separate by column chromatography to obtain 4-(4-pyridyl)-biphenyl-4'-ethanone; The reaction formula is as follows: ； Step 2: Dissolve the 4-(4-pyridyl)-biphenyl-4'-ethanone and malononitrile prepared in step 1 in toluene, add ammonium acetate and acetic acid, stir and heat under reflux, cool, and separate by column chromatography to obtain 2-isocyano-3-[4-(4-pyridyl)biphenyl-4'-yl]but-2-enenitrile; The reaction formula is as follows: ； Step 3: Potassium tert-butoxide and (formylmethylene)triphenylphosphine were stirred in a toluene solvent on an ice bath, added to 6-methoxy-2-naphthaldehyde, heated under reflux under nitrogen protection, cooled, and separated by column chromatography to obtain 3-(6-methoxynaphth-2-yl)propenal; The reaction formula is as follows: ； Step 4: dissolving the 2-isocyano-3-[4-(4-pyridinyl)biphenyl-4'-yl]but-2-enenitrile prepared in step 2, the 3-(6-methoxynaphthalen-2-yl)acrolein prepared in step 3, and piperidine in dichloromethane, stirring and heating under reflux, cooling, and separating by column chromatography to obtain 2-isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile; The reaction formula is as follows: ； Step 5: dissolving the 2-isocyanato-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile and iodomethane obtained in step 4 in acetonitrile, stirring and heating under reflux, cooling and filtering, and eluting with anhydrous ether to obtain the hemoglobin fluorescent probe; The reaction formula is as follows: 。 3. The method for preparing a hemoglobin fluorescent probe according to claim 2, wherein: The specific process of step 1 is: 1.1 Mix 4-pyridineboric acid, tetrakis(triphenylphosphine)palladium and K2CO3 with 1,4-dioxane and H2O, and stir to form a mixed solution; 1.2 Dissolve 4′-(4-bromophenyl)acetophenone in 1,4-dioxane and mix with the mixture from step 1.

1. Reflux the mixture under nitrogen protection for 8 hours. Monitor the reaction progress by TLC until the starting material 4′-(4-bromophenyl)acetophenone is completely consumed. 1.3 After the reaction is completed, the mixture is cooled to room temperature, and the reactant is extracted three times with water and dichloromethane. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product is purified on a silica gel column to obtain 4-(4-pyridyl)-biphenyl-4'-ethanone; The molar ratio of 4-pyridineboric acid, tetrakis(triphenylphosphine)palladium, K2CO3 and 4′-(4-bromophenyl)acetophenone is 3:0.05:6:2; the volume ratio of 1,4-dioxane and H2O is 4:1, wherein the molar volume ratio of tetrakis(triphenylphosphine)palladium to 1,4-dioxane is 3.1-3.2 mmol / L.

4. The method for preparing a hemoglobin fluorescent probe according to claim 2, wherein: The specific synthesis process of step 2 is: 4-(4-pyridyl)-biphenyl-4'-ethanone and malononitrile were dissolved in toluene, and then ammonium acetate and acetic acid were added and refluxed for 12 hours. After the reaction was completed as monitored by TLC, the mixture was cooled to room temperature and extracted with water and dichloromethane. After concentration under reduced pressure, the crude product was purified on a silica gel column to obtain 2-isocyano-3-[4-(4-pyridyl)biphenyl-4'-yl]but-2-enenitrile. The molar ratio of 4-(4-pyridyl)-biphenyl-4'-ethanone, malononitrile, ammonium acetate and acetic acid is 14:15.4:4.62:13.

02.

5. The method for preparing a hemoglobin fluorescent probe according to claim 2, wherein: The specific synthesis process of step 3 is: Potassium tert-butoxide and (formylmethylene)triphenylphosphine were stirred in toluene in an ice bath for 1 to 2 hours, then slowly added dropwise to 6-methoxy-2-naphthaldehyde, mixed, protected by nitrogen, and refluxed for 24 hours. After the reaction, the reaction was quenched with 30% dilute hydrochloric acid, and then extracted with water and ethyl acetate. After evaporation of the solvent, the crude product was purified on a silica gel column to obtain 3-(6-methoxynaphthalen-2-yl)propenal. The molar ratio of 6-methoxy-2-naphthaldehyde, potassium tert-butoxide, and (formylmethylene)triphenylphosphine is 3:6:3.

3.

6. The method for preparing a hemoglobin fluorescent probe according to claim 2, wherein: The specific synthesis process of step 4 is: 2-Isocyano-3-[4-(4-pyridinyl)biphenyl-4'-yl]but-2-enenitrile and 3-(6-methoxynaphthalen-2-yl)acrolein in a molar ratio of 0.9:1 were mixed, dissolved in dichloromethane with piperidine, and refluxed for 6 to 8 hours. After concentration under reduced pressure, the crude product was purified on a silica gel column to obtain 2-isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile.

7. The method for preparing a hemoglobin fluorescent probe according to claim 2, wherein: The specific synthesis process of step 5 is: 2-Isocyano-7-(6-methoxynaphthalen-2-yl)-3-[4'-(pyridin-4-yl)biphenyl-4-yl]hept-2,4,6-trienenitrile and iodomethane in a molar ratio of 0.43:1.3 were dissolved in acetonitrile and refluxed for 24 h. After concentration under reduced pressure, acetonitrile was added until dissolved, and the mixture was precipitated with diethyl ether. The mixture was filtered and washed with diethyl ether to obtain the target hemoglobin fluorescent probe.

8. Use of the hemoglobin fluorescent probe according to claim 1 in hemoglobin detection or hemoglobin-mediated controllable intracellular fluorescence imaging, wherein the use is for non-diagnostic or non-therapeutic purposes.

Images