A fluorescent probe targeting the Golgi apparatus to monitor polarity and its preparation and application

Abstract

The present invention belongs to the technical field of fluorescent probes, and specifically relates to a fluorescent probe that targets the Golgi apparatus to monitor polarity, and its preparation and application. The structural formula of the fluorescent probe of the present invention is: the probe uses sulfonamide as a Golgi targeting group and a coumarin 343 fragment as a polarity-specific group to form a typical D-π-A structure, and the GCSP has the ability to detect changes in microenvironment polarity through the intramolecular charge transfer effect. The probe of the present invention can monitor the polarity changes of the Golgi apparatus in living cells in real time, and for the first time realizes the visualization of the polarity of the Golgi apparatus in liver adipose tissue and cancer cell tissue, revealing that the polarity of the Golgi apparatus in fatty liver and cancer is low, and successfully confirming that the polarity of the Golgi apparatus can be used as a marker for early diagnosis of cancer, and provides a powerful tool for revealing the connection between the polarity of the Golgi apparatus and serious diseases in future clinical studies.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe targeting a Golgi apparatus for monitoring polarity, and preparation and application thereof. Background Art

[0002] Polarity is an important parameter in living systems because many metabolic reaction processes strongly depend on the surrounding polarity. Many life activities, including oxidative damage, membrane permeability, protein transport, and cell division, can lead to fluctuations in polarity. Abnormal changes in polarity are closely related to diseases such as cirrhosis, diabetes, and depression.

[0003] The Golgi apparatus is an important type of intracellular organelle, mainly composed of three basic units: flat sacs, large vesicles, and small vesicles. Its main function is to modify, package, and classify proteins transported by the endoplasmic reticulum, and then transport them to specific parts of the cell or secrete them outside the cell. The polarity level of the Golgi apparatus can reflect its basic state and function, and plays a key role in regulating life activities. Abnormal polarity levels of the Golgi apparatus often disrupt protein metabolism, leading to Golgi apparatus dysfunction and even causing many diseases. Therefore, real-time tracking and monitoring of fluctuations in Golgi polarity in living cells is extremely important for the early diagnosis and pathological research of Golgi polarity-related diseases.

[0004] Fluorescence imaging is an effective technical means to detect various bioactive substances in biological systems in situ in a non-invasive, highly sensitive and easy-to-operate manner. Although a large number of fluorescent probes have been reported for Golgi-specific imaging, to our knowledge, only three probes are polarity sensitive. Due to the lack of Golgi-specific polarity fluorescent probes and various disease models, the intrinsic relationship between related diseases and Golgi polarity has not been clearly elucidated. At present, disease models using Golgi polarity are mainly focused on the early diagnosis of depression and renal injury at the cellular or animal tissue level. This limited disease model hinders our in-depth understanding of the relationship between Golgi polarity and related diseases. To our knowledge, there is no Golgi-specific polarity fluorescent probe that can realize the visualization of cancer models (such as clinical cancer patient samples) and Golgi polarity fluctuations. In addition, the practical application of existing Golgi-specific polarity probes is limited by shortcomings such as complex structure, cumbersome synthesis, low sensitivity, and small Stokes shift. Therefore, we hoped to design a novel Golgi-specific polarity fluorescent probe with high sensitivity and selectivity for polarity for early diagnosis and fluorescence imaging studies of fatty liver and cancer models. Summary of the invention

[0005] In view of the problems existing in the current prior art, the present invention provides a fluorescent probe targeting the Golgi apparatus to monitor polarity and the preparation and application thereof.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a fluorescent probe targeting the Golgi apparatus to monitor polarity, wherein the structural formula of the fluorescent probe is:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the aforementioned fluorescent probe targeting the Golgi apparatus to monitor polarity, the preparation method comprising the following steps:

[0010] Step 1: After mixing coumarin 343 and sulfonamide in dimethylformamide, add N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, heat to reflux under nitrogen protection, and add water to quench the reaction to obtain a reaction solution:

[0011] Step 2: extracting the reaction solution, and drying and concentrating the organic phase obtained by extraction to obtain a crude product;

[0012] Step 3: Using acetonitrile / water as eluent, purify the crude product by C18 chromatographic column to obtain the fluorescent probe GCSP.

[0013] Preferably, the molar ratio of coumarin 343 to sulfonamide is 1:1-1:1.2.

[0014] Preferably, the heating reflux temperature is 105-115° C. and the time is 1.8-2.25 hours.

[0015] Preferably, the mass ratio of N,N-diisopropylethylamine to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.4-1:1.7.

[0016] Preferably, the specific process of step 2 is: extracting the reaction solution with ethyl acetate, washing the obtained organic phase with brine, then drying and filtering with Na2SO4, and concentrating the obtained filtrate by reduced pressure distillation to obtain a crude product.

[0017] In a third aspect, the present invention provides the use of the aforementioned fluorescent probe targeting the Golgi apparatus to monitor polarity in detecting the polarity of the Golgi apparatus.

[0018] In a fourth aspect, the present invention provides the use of the aforementioned fluorescent probe targeting the Golgi apparatus for monitoring polarity in the preparation of a reagent for detecting changes in the polarity of the Golgi apparatus.

[0019] In a fifth aspect, the present invention provides the use of the aforementioned fluorescent probe targeting the Golgi apparatus for monitoring polarity in the preparation of a reagent for early diagnosis of fatty liver.

[0020] In a sixth aspect, the present invention provides the use of the aforementioned fluorescent probe targeting the Golgi apparatus to monitor polarity in the preparation of a reagent for identifying cancer cells.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The fluorescent probe GCSP of the present invention uses sulfonamide as an electron donor (D) and a Golgi targeting group, and coumarin 343 as an electron acceptor (A) and a polarity-specific group to form a typical D-π-A structure. Through the intramolecular charge transfer (ICT) effect, GCSP has the ability to detect changes in microenvironment polarity. Its detection mechanism is: under low-polarity conditions, the probe GCSP has less charge separation and weak interaction with the solvent, and the dipole moment does not change much, emitting short and strong fluorescence. As the polarity increases, due to the dipole-dipole interaction between the probe molecule and the solvent, GCSP will produce a larger charge separation, and the excited state energy will be rapidly released through non-radiative transitions, thereby weakening the fluorescence intensity and realizing the detection of the polarity of the microenvironment.

[0023] (2) The fluorescent probe GCSP of the present invention is the first to use the coumarin 343 fragment as a polarity-specific group to synthesize a Golgi apparatus polar fluorescent probe.

[0024] (3) The fluorescent probe GCSP of the present invention has high sensitivity and selectivity for detecting changes in microenvironment polarity and is not interfered by microenvironment viscosity, pH changes, other biological analytes (including reactive oxygen species, cations, anions, thiols and amino acids), etc.

[0025] (4) The fluorescent probe GCSP of the present invention has good specificity, targets and labels the Golgi apparatus, and can sensitively monitor changes in the polarity of the Golgi apparatus microenvironment.

[0026] (5) The fluorescent probe GCSP of the present invention is combined with laser confocal microscopy imaging technology to realize the visualization imaging of the changes in Golgi polarity in cancerous cells, mouse fatty liver model tissue sections, mouse tumor model tissue sections, and clinical patient cancer tissue sections. It is the first time to reveal that the polarity of the Golgi apparatus is extremely low under fatty liver conditions and that the polarity of the Golgi apparatus can be used to distinguish normal cells from cancer cells.

[0027] (6) The method of the fluorescent probe GCSP targeting the Golgi apparatus to monitor polarity is simple, rapid and sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For the NMR characterization of GCSP of the present invention, 1H-NMR and 13 C-NMR spectrum.

[0029] Figure 2 This is the mass spectrometry characterization of the GCSP of the present invention, HR-MS spectrum.

[0030] Figure 3 The fluorescence spectra and ultraviolet absorption spectra of the GCSP of the present invention in solvents of different polarities are shown in FIG.

[0031] Figure 4 The fluorescence spectra of GCSP of the present invention in tetrahydrofuran and methanol / glycerol system with different glycerol contents.

[0032] Figure 5 The fluorescence spectra of GCSP of the present invention in tetrahydrofuran and water systems with different pH values ​​are shown in FIG.

[0033] Figure 6 The selectivity of the GCSP of the present invention to polarity and small molecules such as common cations, anions and amino acids in a water system with a pH of 7.4.

[0034] Figure 7 It is the fluorescence stability of GCSP of the present invention in tetrahydrofuran or aqueous solution.

[0035] Figure 8 It is a co-localization imaging diagram of the GCSP of the present invention and the commercial Golgi apparatus specific dye NBD C6-Ceramide (NBD), the lysosome specific dye Lyso-Brite NIR (LB-NIR) and the mitochondria specific dye Mito-Tracker Deep Red (MTDR).

[0036] Fig. 9 This is the fluorescence imaging diagram of GCSP in normal liver tissue and fatty liver tissue of the present invention.

[0037] Fig.10 The fluorescence imaging diagrams of the GCSP of the present invention and three cancer cells (HeLa, A549, HepG2) and two normal cells (PC12, MPC5).

[0038] Fig.11 This is the fluorescence imaging diagram of the GCSP of the present invention on normal tissue slices and tumor tissue slices of a tumor-bearing mouse model.

[0039] Fig.12 The GCSP of the present invention is a fluorescence imaging diagram of normal tissue slices and cancerous tissue slices of clinical cancer patients. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] Example 1

[0042] Preparation and characterization of GCSP:

[0043]

[0044] In a round-bottom flask, dimethylformamide (DMF) (3 mL), coumarin 343 (300 mg, 1.05 mmol) and sulfonamide (217 mg, 1.26 mmol) were added, and after mixing, N,N-diisopropylethylamine (DIEA) (407.7 mg) and N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (599.7 mg) were added, and under nitrogen protection, the mixture was refluxed at 110 ° C for 2 hours, and then water (3 mL) was added to quench the reaction to obtain a reaction solution;

[0045] The reaction solution was extracted with ethyl acetate, and the obtained organic phase was washed with brine, then dried and filtered with Na2SO4, and the obtained filtrate was concentrated by vacuum distillation. The obtained crude product was dissolved in methanol (2 mL) and eluted with a C18 chromatographic column in a 5-80% acetonitrile water system for 50 min to obtain a yellow solid, which was the fluorescent probe GCSP (22 mg, 4.29%).

[0046] 1 H-NMR(600MHz,DMSO-d6)δ11.03-10.97(d,1H),8.64-8.63(d,1H),7.88-7.72(m,4H) ,7.34-7.28(d,2H),7.07(s,1H),2.84(s,4H),1.93-1.85(m,4H),1.13-1.10(m,4H). 13 C-NMR(150MHz,DMSO-d6)δ162.82,161.94,159.20,152.82,151.60,148.59,146.71,142.19,128.01,127 .34,126.08,119.75,118.99,108.19,107.40,105.80,105.21,49.81,27.31,21.29,20.38,20.19.HR-MS m / z:[MH] - Calculated for C22 H 20 N3O5S,438.1202; measured,438.1131.

[0047] Example 2

[0048] The GCSP in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1.0 mM mother solution. The polarity response test solution, i.e., dioxane, ethyl acetate, tetrahydrofuran (THF), benzonitrile, acetonitrile and dimethyl sulfoxide (DMSO), were all spectrally pure. The excitation wavelength was fixed at 440 nm, and the excitation / emission slit width was 0.5 nm. The fluorescence spectra and ultraviolet absorption spectra of GCSP with a final concentration of 1 μM and 5 μM in different polar solutions were recorded using a fluorescence spectrometer and a UV-visible absorption spectrometer, respectively. Figure 3 ). As the polarity of the solvent increases, the UV absorption spectrum hardly changes, and the fluorescence spectrum undergoes an obvious red shift, indicating that the probe has typical intramolecular charge transfer characteristics.

[0049] Example 3

[0050] The GCSP concentration in Example 1 was kept at 1 μM, and the GCSP concentrations at different viscosities ( Figure 4 )、pH( Figure 5 ), common ions and amino acids ( Figure 6 ) in the presence of polarity. The results show that the probe's selectivity for polarity is much greater than viscosity, pH, and other biological analytes (including reactive oxygen species, cations, anions, thiols, and amino acids). In addition, the fluorescence intensity of the probe in THF or water changes very little over time ( Figure 7 ), indicating that the probe has good photostability. The above results are conducive to the detection of polarity fluctuations in complex biological systems. The order and concentration of the substances are: 1. Blank; 2, THF; 3, Pro; 4, Hcy; 5, GSH; 6, Cys; 7, Ala; 8, Phe; 9, Val; 10, Ser; 11, Ile; 12, Lys; 13, His; 14, Thr; 15, Tyr; 16, Gln; 17, Leu; 18, Asn; 19, Arg; 20, Glu; 21, Cu 2+ ; 22,Fe 2+ ; 23,S 2- ; 24,K + ; 25, H2O2; 26, Na + ;27,Li + ; 28, NO2 - ; 29,Fe 3+ ; 30,CO3 2- ; 31,Ba 2+ ; 32, NO3- ; 33, SO4 2- ; 34, Cl-; 35, Br - ; 36, S2O3 2- ; 37,HSO3 - ; 38,H2PO4 - ; 39,CN-.

[0051] Example 4

[0052] In order to investigate the Golgi targeting of GCSP in Example 1, this example first conducted a co-localization experiment of GCSP with the Golgi green fluorescent dye NBD C6-Ceramide (NBD), the lysosomal red fluorescent dye Lyso-Brite NIR (LB-NIR), and the mitochondrial red fluorescent dye Mito-Tracker Deep Red (MTDR). After GCSP (3.0 μM) and NBD (5.0 μM), LB-NIR (0.3 μM) or MTDR (0.3 μM) were co-incubated in adherent human cervical cancer cells (HeLa) for 30 minutes, the co-localization of the two was observed under a laser confocal microscope. Among them, the fixed excitation wavelength of GCSP was 405 nm, and the green fluorescence emission range of 430-500 nm was collected; the fixed excitation wavelength of NBD was 488 nm, and the green fluorescence emission range of 500-560 nm was collected; the fixed excitation wavelength of LB-NIR and MTDR was 633 nm, and the red fluorescence emission range of 650-750 nm was collected. The results are as follows: Figure 8 As shown, GCSP overlaps well with NBD ( Figure 8 b, c), and the Pearson colocalization coefficient PC in HeLa cells was 0.85 ( Figure 8 d). In contrast, GCSP exhibited poor fluorescence overlap with LB-NIR or MTDR in HeLa cells, with PC values ​​of only 0.49 ( Figure 8 h) or 0.40( Figure 8 l), confirming that GCSP has excellent Golgi targeting ability.

[0053] Example 5

[0054] A fatty liver mouse model was successfully established by feeding with a high-fat diet and injecting dexamethasone. The normal liver and fatty liver were separated and sliced ​​at a thickness of 4 μm, and then GCSP (20 μM) was added for incubation. The GCSP excitation wavelength was fixed at 405 nm, and the green fluorescence emission range of 430-490 nm was collected. The imaging was observed under a laser confocal microscope. Fatty liver tissue showed obvious fluorescence signals, while normal liver tissue had almost no fluorescence emission ( Fig. 9), which revealed for the first time that the polarity level of Golgi apparatus was low under fatty liver conditions. In addition, hematoxylin-eosin (H&E) staining was used to verify the fatty liver model. Fig. 9 f shows that the healthy liver tissue structure and cell morphology are normal, and the hepatic cords are arranged regularly. However, in the fatty liver model group, the hepatocytes were swollen and vacuolated in large numbers, and the hepatic cords were disordered ( Fig. 9 g), severe fatty lesions. These results indicate that GCSP can achieve early diagnosis of fatty liver by fluorescence imaging of Golgi polarity.

[0055] Example 6

[0056] The GCSP (3.0 μM) in Example 1 was used to incubate cancer cells HeLa, A549, HepG2 and normal cells PC12 and MPC5. The GCSP excitation wavelength was fixed at 405 nm, and the green fluorescence emission range of 430-490 nm was collected. The imaging was observed under a laser confocal microscope ( Fig.10 ), compared with the negligible fluorescence signal of normal cells, the fluorescence signals of all cancer cells were significantly enhanced, indicating that the polarity of the Golgi apparatus was reduced in tumors. This shows that GCSP can distinguish cancer cells from normal cells by detecting the polarity of the Golgi apparatus.

[0057] Example 7

[0058] In order to investigate the potential of GCSP in diagnosing cancer at the tissue section level in Example 1, a tumor-bearing mouse model was prepared by subcutaneously injecting H22 cells into the right axilla of mice for about 14 days. In the mouse tissue section imaging, normal tissue and tumor tissue were sliced ​​into 4 μm thickness, incubated with GCSP (20 μM) for 30 min, washed 3 times with PBS (pH = 7.4), and finally imaged with a laser confocal microscope (λ ex =405nm,λ em =430-490nm) for fluorescence imaging. Fig.11 As shown, tumor tissue ( Fig.11 f) has a higher fluorescence than other normal tissues ( Fig.11 ae) was significantly enhanced, further confirming that GCSP can efficiently diagnose cancer.

[0059] Example 8

[0060] In this example, three normal / cancerous tissues (intestine, thyroid and breast) collected from surgical specimens of patients were frozen and sliced ​​into 4 μm sections, incubated with GCSP (20 μM) for 30 min, and imaged using a laser confocal microscope (λ ex =405nm,λ em =430~490nm) for fluorescence imaging. To our delight, we found that the fluorescence of all cancer tissues was significantly stronger than that of normal tissues ( Fig.12af,s), which is consistent with the results of H&E staining ( Fig.12 mr), indicating that GCSP has clinical diagnostic potential for human cancer.

[0061] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a fluorescent probe targeting the Golgi apparatus to monitor polarity in the preparation of a Golgi apparatus polarity change detection reagent and / or a fatty liver early diagnosis reagent and / or a cancer cell identification reagent, characterized in that: The structural formula of the fluorescent probe is: 。 2. The use of the fluorescent probe targeting the Golgi apparatus to monitor polarity according to claim 1, characterized in that: The preparation method of the fluorescent probe comprises the following steps: Step 1: After mixing coumarin 343 and sulfonamide in dimethylformamide, add N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, heat to reflux under nitrogen protection, and add water to quench the reaction to obtain a reaction solution: Step 2: extracting the reaction solution, and drying and concentrating the organic phase obtained by extraction to obtain a crude product; Step 3: Using acetonitrile / water as eluent, purify the crude product by C18 chromatographic column to obtain the fluorescent probe GCSP.

3. The use of the fluorescent probe targeting the Golgi apparatus to monitor polarity according to claim 2, characterized in that: The molar ratio of coumarin 343 to sulfonamide is 1:1-1:1.

2.

4. The use of the fluorescent probe targeting the Golgi apparatus to monitor polarity according to claim 2, characterized in that: The heating reflux temperature is 105-115° C. and the time is 1.8-2.25 hours.

5. The use of the fluorescent probe targeting the Golgi apparatus to monitor polarity according to claim 2, characterized in that: The mass ratio of the N,N-diisopropylethylamine to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.4-1:1.

7.

6. The use of the fluorescent probe targeting the Golgi apparatus to monitor polarity according to claim 2, characterized in that: The specific process of step 2 is: extracting the reaction solution with ethyl acetate, washing the obtained organic phase with brine, then drying and filtering with Na2SO4, and concentrating the obtained filtrate by reduced pressure distillation to obtain a crude product.

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