A large Stokes shift near-infrared fluorescence probe for detecting HClO, its preparation method and biological imaging application

Abstract

The present invention provides a large Stokes shift near-infrared fluorescent probe for detecting HClO, its preparation method and biological imaging application, belonging to the field of bioanalysis technology. Based on the strategy of modifying the substituents at the 3,6 positions of xanthene, the present invention constructs a large Stokes shift near-infrared fluorescent probe for specifically recognizing HClO. Optical experiments show that the emission wavelength of the probe for detecting HClO is 776 nm, the Stokes shift reaches 148 nm, with strong selectivity, high sensitivity, and a detection limit as low as 4.95 nM, which can greatly reduce the interference of background fluorescence in biological imaging applications. The fluorescent probe of the present invention has almost no cytotoxicity and can be applied to the detection and imaging of HClO in living cells MCF-7 and rat liver tissues, with a tissue penetration depth of 140 μm.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis, and particularly relates to a large Stokes shift near-infrared fluorescent probe for detecting HClO, a preparation method thereof, and a biological imaging application. Background Art

[0002] Hypochlorous acid (HClO) is one of the important reactive oxygen species (ROS) in biology. It plays a crucial role in the immune defense against pathogens and contributes to host defense. In addition, HClO is widely used as a disinfectant in our daily life. A large number of studies have shown that excessive HClO can cause oxidative stress and oxidative damage to biomolecules such as nucleic acids, proteins, and lipids, thereby leading to the occurrence of many diseases, such as Parkinson's disease, inflammatory diseases, atherosclerosis, cardiovascular diseases, etc. Therefore, in view of the biological importance of HClO, studying the changes in the concentration of HClO in the living system is of great significance for biological research and early disease diagnosis. Currently, there are many methods available for selectively detecting hypochlorous acid / hypochlorite, such as iodometric titration, colorimetry, chemiluminescence, etc. However, these methods are often cumbersome, costly, and have low sensitivity and specificity, which limits their wide application. Compared with the above methods, fluorescent probes have the advantages of high selectivity and sensitivity, wide detection range, fast response speed, and little damage to samples, and are widely used in biological detection.

[0003] It is reported that scientific researchers have developed some fluorescent probes for detecting HClO. Among them, xanthene dyes are widely used in the construction of fluorescent probes due to their excellent photophysical properties (including large molar extinction coefficient, high fluorescence quantum yield and good photostability). At present, most of the probes for detecting HClO focus on the visible light region (Ren M, Li Z, Deng B, et al. Single fluorescent probe separately and continuously visualize H2S and HClO in lysosomes with different fluorescence signals[J]. Analytical chemistry, 2019, 91(4):2932-2938. Yuan L, Lin W, Xie Y, et al. Fluorescent detection of hypochlorous acid from turn-on to FRET-based ratiometry by a HOCl-mediated cyclization reaction[J]. Chemistry, 2012, 18(09):2700-2706.). Although there are some near-infrared fluorescent probes, their Stokes shift is small (Zhang C, Nie Q, Ismail I, et al. A highly sensitive and selective fluorescent probe for fast sensing of endogenous HClO in living cells[J]. Chemical Communications, 2018, 54(31):3835-3838.). The short emission wavelength limits their penetration depth in organisms, and the severe crosstalk between excitation and emission leads to serious self-absorption of fluorescence, which all limit their applications in biology and medicine. In view of the above situation, it is of great research significance and application value to develop fluorescent probes with near-infrared emission and large Stokes shift to monitor the HClO level in living cells and live animals. Summary of the Invention

[0004] To solve the technical problems existing in the above-mentioned fluorescence probes for detecting HClO, the present invention proposes a large Stokes shift near-infrared fluorescence probe for detecting HClO, its preparation method and biological imaging application. Based on the strategy of modifying the substituents at the 3,6 positions of xanthene, an asymmetric skeleton fluorescence probe RHSBZ with an enlarged conjugated structure is constructed. This probe has near-infrared luminescence characteristics, a Stokes shift of up to 148 nm, can quantitatively detect HClO, has good selectivity, high sensitivity, a detection limit as low as 4.95 nM, can be successfully applied to imaging MCF-7 cells and rat liver tissues, and the tissue penetration depth reaches 140 μm, providing an important approach for the diagnosis and treatment of related diseases.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention: A large Stokes shift near-infrared fluorescence probe (RHSBZ) for detecting HClO, the molecular formula of the fluorescence probe is C 38 H 33 N3O2S2, and the structural formula is:

[0007]

[0008] Another technical solution of the present invention: A preparation method of a large Stokes shift near-infrared fluorescence probe for detecting HClO, and the synthesis route is as follows:

[0009]

[0010] Specifically, it includes the following steps:

[0011] Dissolve 2-methoxyphenothiazine and sodium hydroxide in dimethyl sulfoxide, add iodoethane, and react at 65 °C for 9 h under a protective atmosphere to obtain reaction solution 1. Extract, wash, dry, and distill under reduced pressure the reaction solution 1, and separate and purify it by column chromatography to obtain intermediate M1;

[0012] Dissolve the intermediate M1 and 4-diethylaminoketonic acid in methanesulfonic acid, react at 70 °C for 12 h to obtain reaction solution 2, adjust the pH of the reaction solution 2 to 7-8 and stir, then extract, dry, distill under reduced pressure, and separate and purify it by column chromatography to obtain intermediate M2;

[0013] Add the intermediate M2, o-aminophenol, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide to dichloromethane and stir overnight to obtain reaction solution 3. Wash, dry, distill under reduced pressure the reaction solution 3, and separate and purify it by column chromatography to obtain intermediate M3;

[0014] The intermediate M3 and Lawesson's reagent are added to toluene, and under a protective atmosphere, the mixture is stirred at 90 °C for 4 h to obtain reaction solution 4. The reaction solution 4 is distilled under reduced pressure, and separated and purified by column chromatography to obtain the large Stokes shift fluorescent probe (fluorescent probe RHSBZ).

[0015] More specifically, it includes the following steps:

[0016] 2-Methoxyphenothiazine and crushed sodium hydroxide are dissolved in dimethyl sulfoxide (DMSO), and ethyl iodide is added. The reaction is carried out at 65 °C for 9 h under a nitrogen atmosphere to obtain reaction solution 1. The reaction solution 1 is cooled and poured into water, extracted three times with dichloromethane, then the combined organic phases are washed with saturated sodium chloride solution and dried with anhydrous sodium sulfate, and distilled under reduced pressure to obtain a crude product. The obtained crude product is separated and purified by gradient elution. The ratio range of petroleum ether - dichloromethane in the eluent used is 100:1 - 10:1 (v / v), and a white solid is obtained, which is intermediate M1;

[0017] Intermediate M1 and 4-diethylaminoketone acid are dissolved in methanesulfonic acid, and the reaction is carried out at 70 °C for 12 h to obtain reaction solution 2. The reaction solution 2 is poured into stirred ice water, the pH is adjusted to 7 - 8 with an aqueous sodium bicarbonate solution and stirred for 20 min, then extracted three times with dichloromethane. The organic layer is dried with anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product. The crude product is separated and purified by gradient elution. The ratio range of petroleum ether - dichloromethane in the eluent used is 100:1 - 10:1 (v / v), and a blue solid is obtained, which is intermediate M2;

[0018] Intermediate M2, o-aminophenol, DMAP (4-dimethylaminopyridine), and DCC (dicyclohexylcarbodiimide) are added to dry dichloromethane, and stirred at room temperature overnight to obtain reaction solution 3. After the reaction solution 3 is washed three times with water, it is dried with anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product. The crude product is separated and purified with column chromatography silica gel. The eluent used is a mixture of petroleum ether - ethyl acetate (100:1 - 10:1, v / v), and a green solid is obtained, which is intermediate M3;

[0019] Intermediate M3 and Lawesson's reagent are added to dry toluene, and under a nitrogen atmosphere, the mixture is stirred at 90 °C for 4 h to obtain reaction solution 4. The reaction solution 4 is distilled under reduced pressure to obtain a crude product. The crude product is separated and purified by gradient elution. The ratio range of petroleum ether - dichloromethane in the eluent used is 100:1 - 10:1 (v / v), and a yellow solid is obtained, which is the large Stokes shift fluorescent probe (fluorescent probe RHSBZ).

[0020] Further, the molar ratio of 2-methoxyphenothiazine, sodium hydroxide and iodoethane is 1:2:2.5, and the volume ratio of iodoethane to dimethyl sulfoxide is 1:10.

[0021] Further, the molar ratio of intermediate M1 to 4-diethylamino keto acid is 1:1.

[0022] Further, the molar ratio of intermediate M2, o-aminophenol, 4-dimethylaminopyridine to dicyclohexylcarbodiimide is 1:1.1:1:2.5.

[0023] Further, the molar ratio of intermediate M3 to Lawesson's reagent is 1:1.08.

[0024] The third technical solution of the present invention: the application of the large Stokes shift fluorescent probe in detecting HClO, the lowest detection limit of HClO is 4.95 nM, and the concentration of the fluorescent probe is 10 μM. HClO can oxidize the S atom on the spiro ring of the fluorescent probe and form an electron-deficient center on the carbonyl carbon to obtain an intermediate. The intermediate is immediately nucleophilically attacked by the adjacent phenolic hydroxyl group, and at the same time, the leaving of SCl - to obtain the final cyclized product benzoxazole, generating a compound with fluorescent properties, realizing the detection of HClO by the fluorescent probe.

[0025] Further, when detecting HClO, the fluorescent probe needs to be dissolved in a buffer solution, the reaction temperature is 37 °C, and the pH of the incubation system is 5-10.

[0026] Furthermore, the buffer solution is an EtOH / PBS (1:1 v / v, 5 mM) system, preferably the reaction temperature is 37 °C, and the pH of the incubation system is 7.4.

[0027] Further, a steady-state / transient fluorescence spectrometer is used to detect the probe RHSBZ, and the quantitative fluorescence detection parameters are: the excitation wavelength is 610 nm, and the data collection range of the emission spectrum is 630-900 nm. The excitation slit width is 7.5 nm, and the emission slit width is 7 nm.

[0028] Further, it is used for detecting and imaging the concentration level of HClO in living cells or tissues.

[0029] Furthermore, the live cell imaging experiment is carried out under a STELLARIS 5 super-resolution laser confocal microscope using a 633 nm laser. Among them, the cells are tumor cells, and more specifically, breast cancer cells MCF-7.

[0030] Furthermore, tissue imaging was performed under a Zeiss LSM 880 confocal laser scanning microscope, and images were collected at an excitation wavelength of 633 nm, where the tissue was a rat liver section.

[0031] Compared with the prior art, the present invention has the following advantages and technical effects:

[0032] 1. The present invention designed and synthesized a large Stokes shift near-infrared fluorescent probe RHSBZ for detecting HClO. The Stokes shift of the fluorescent probe RHSBZ reaches 148 nm, effectively reducing background interference.

[0033] 2. The concentration of the fluorescent probe prepared in the present invention for detecting HClO is 10 μM. The fluorescence intensity of the fluorescent probe in the present invention at the maximum emission peak has a good linear relationship with the concentration of HClO in the range of 0 - 5 μM, and its detection limit is calculated to be as low as 4.95 nM, indicating that the fluorescent probe RHSBZ prepared in the present invention can be used as a chemical sensor to quantitatively detect HClO.

[0034] 3. The maximum emission wavelength of the fluorescent probe prepared in the present invention is 776 nm, which is in the near-infrared region, has strong penetrability, causes little damage to biological samples, and has good stability. The fluorescence intensity remains stable in a wide pH range such as acidic, neutral, and alkaline.

[0035] 4. The fluorescent probe of the present invention is not interfered by other competitive ROS / amino acids / metal ions during the detection process, so that the fluorescent probe RHSBZ has high selectivity and good anti-interference ability for the detection of HClO.

[0036] 5. The fluorescent probe of the present invention has a rapid response time. Fluorescence appears within 5 s after adding HClO, and the fluorescence intensity increases significantly with time. The fluorescence intensity reaches the maximum at about 100 s and then remains stable, indicating that the fluorescent probe RHSBZ prepared in the present invention will provide a rapid analysis method for detecting HClO.

[0037] 6. The fluorescent probe provided by the present invention has almost no toxicity to cells and can be widely used for the detection and imaging of HClO in living cells and tissues. The tissue penetration depth reaches 140 μm, having good practicability.

[0038] 7. The preparation method of the present invention is simple, the raw materials are cheap and easily available, and it is easy to be popularized in industrialization, having good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application. In the drawings:

[0040] Figure 1 Molecular structure diagram of the fluorescent probe RHSBZ prepared in Example 1;

[0041] Figure 2 HNMR spectrum of the fluorescent probe RHSBZ prepared in Example 1;

[0042] Figure 3 CNMR spectrum of the fluorescent probe RHSBZ prepared in Example 1;

[0043] Figure 4 MS spectrum of the fluorescent probe RHSBZ prepared in Example 1;

[0044] Figure 5 UV-Vis absorption spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 before and after responding to HClO (20 μM) in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system;

[0045] Figure 6 Fluorescence spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 before and after responding to HClO (20 μM) in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system;

[0046] Figure 7 Normalized graphs of absorption and fluorescence spectra of the fluorescent probe RHSBZ (10 μM) of the present invention after responding to HClO (20 μM) in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system;

[0047] Figure 8 UV-Vis titration spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 with gradually added 0 - 20 μM HClO in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system;

[0048] Figure 9 Fluorescence emission titration spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 with gradually added 0 - 20 μM HClO in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system;

[0049] Figure 10 Linear relationship graph between the emission intensity of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 at 776 nm and the HClO concentration (0 - 5 μM);

[0050] Figure 11The UV absorption spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system after adding HClO (20 μM) and 16 other reactive oxygen species / bio-thiols / metal ions (ONOO - , H2O2, ·OH, ROO·, t-BuOO·, 1 O2, t-BuOOH, NO2 - , GSH, Cys, Hcy, Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ );

[0051] Figure 12 The fluorescence emission spectra of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system after adding HClO (20 μM) and 16 other reactive oxygen species / bio-thiols / metal ions (ONOO - , H2O2, ·OH, ROO·, t-BuOO·, 1 O2, t-BuOOH, NO2 - , GSH, Cys, Hcy, Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ );

[0052] Figure 13 The anti-interference experimental graphs of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 against HClO (20 μM) in the presence of various reactive oxygen species / bio-thiols / metal ions (20 μM) (ONOO - , H2O2, ·OH, ROO·, t-BuOO·, 1 O2, t-BuOOH, NO2 - , GSH, Cys, Hcy, Fe 2+ , Co 2+ , Ni 2 + , Cu 2+ , Zn 2+ );

[0053] Figure 14 The change curve of the fluorescence intensity of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 with time in the presence of 20 μM HClO;

[0054] Figure 15Fluorescence emission intensity of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 at 776 nm before and after adding HClO at different pH values;

[0055] Figure 16 Cytotoxicity detection results of the fluorescent probe RHSBZ prepared in Example 1 against MCF-7 cells at different concentrations (0, 1.25, 2.5, 5, 10, 20 μM);

[0056] Figure 17 Fluorescent staining imaging diagram of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 for MCF-7 cells. In the figure, (a1), (b1), (c1) represent the fluorescent staining imaging diagrams of the first group under Bright filed (bright field), Red filed (red channel fluorescence field), and Merged (overlay image) respectively; (a2), (b2), (c2) represent the fluorescent staining imaging diagrams of the second group under Bright filed (bright field), Red filed (red channel fluorescence field), and Merged (overlay image) respectively; (a3), (b3), (c3) represent the fluorescent staining imaging diagrams of the third group under Bright filed (bright field), Red filed (red channel fluorescence field), and Merged (overlay image) respectively; (a4), (b4), (c4) represent the fluorescent staining imaging diagrams of the fourth group under Bright filed (bright field), Red filed (red channel fluorescence field), and Merged (overlay image) respectively;

[0057] Figure 18 Fluorescent imaging diagram of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 in rat liver tissue. Among them, (a) is the fluorescent imaging diagram of group one (first treated with 10 μM of the fluorescent probe RHSBZ prepared in Example 1 and then incubated with 20 μM of HClO in a 37 °C incubator for 60 min, and imaged after washing three times with PBS); (b) is the fluorescent imaging diagram of group two (incubated with 10 μM of the fluorescent probe RHSBZ prepared in Example 1 in a 37 °C incubator for 60 min, and imaged after washing three times with PBS);

[0058] Figure 19 Graph of the change of fluorescence intensity with the scanning depth (at 50 μm); Detailed implementation manners

[0059] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0060] All raw materials used in the embodiments of the present invention are obtained by purchasing commercially.

[0061] The technical solution of the present invention will be further described below through examples.

[0062] Example 1

[0063] 2-Methoxyphenothiazine (2.29 g, 10 mmol) and crushed sodium hydroxide (0.8 g, 20 mmol) were dissolved in 20 mL of dimethyl sulfoxide (DMSO), and iodoethane (2 mL, 25 mmol) was added. The reaction was carried out at 65 °C for 9 h under a nitrogen atmosphere to obtain Reaction Solution 1. Reaction Solution 1 was cooled to 25 °C and poured into water (300 mL). It was extracted three times with dichloromethane, and then the combined organic phase was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The crude product was obtained by distillation under reduced pressure. The crude product was separated and purified by gradient elution using silica gel for column chromatography. The ratio range of petroleum ether - dichloromethane in the eluent used was 100:1 - 10:1 (v / v), and a white solid was obtained, which was Intermediate M1;

[0064] The HNMR spectrum information of Intermediate M1 is as follows: 1 H NMR(400MHz,CDCl3)δ(ppm)7.14(dd,J=7.5,1.2Hz,2H),7.03(d,J=8.4Hz,1H),6.87(d,J=8.0Hz,2H),6.49(d,J=2.2Hz,2H),4.15~3.60(m,5H),1.43(t,J=7.0Hz,3H).

[0065] The CNMR spectrum information of Intermediate M1 is as follows: 13 C NMR(101MHz,CDCl3)δ(ppm)159.88,146.66,144.72,127.42,127.18,125.16,125.10~124.96,122.48,115.33,106.72,104.01,103.26,55.61,41.91,29.83.

[0066] Intermediate M1 (1.03 g, 4 mmol) and 4 - diethylaminoketone acid (1.27 g, 4 mmol) were dissolved in methanesulfonic acid, and the reaction was carried out at 70 °C for 12 h to obtain Reaction Solution 2. Reaction Solution 2 was poured into stirred ice water (100 mL), and the pH was adjusted to 7 - 8 with an aqueous sodium bicarbonate solution and stirred for 20 min. Then it was extracted three times with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The crude product was obtained by distillation under reduced pressure. The crude product was separated and purified by gradient elution. The ratio range of petroleum ether - dichloromethane in the eluent used was 100:1 - 10:1 (v / v), and a blue solid was obtained, which was Intermediate M2;

[0067] The HNMR spectrum information of intermediate M2 is as follows: 1 H NMR(400MHz,CDCl3)δ(ppm)8.00(s,1H),7.70~7.57(m,2H),7.21~7.09(m,2H),7.03(dd,J=7.8,1.4Hz,1H),6.93~6.85(m,2H),6.72(s,1H),6.55(d,J=8.9Hz,1H),6.44(s,2H),6.35(dd,J=8.9,2.2Hz,1H),3.96(q,J=6.9Hz,2H),3.36(q,J=7.1Hz,4H),1.47(t,J=6.9Hz,3H),1.17(t,J=7.0Hz,6H).

[0068] The CNMR spectrum information of intermediate M2 is as follows: 13 C NMR(101MHz,CDCl3)δ(ppm)169.51,152.95,152.68,151.87,149.57,146.94,143.72,134.79,129.56,128.96,127.34,125.78,124.98,124.10,123.74,122.78,118.89,115.26,113.15,108.48,103.03,97.54,49.16,44.54,42.18,33.94,25.63,24.95,12.76,12.51。

[0069] Intermediate M2(0.92g,2mmol),o-aminophenol(0.24g,2.2mmol),DMAP(4-dimethylaminopyridine,0.24g,2mmol) and DCC(dicyclohexylcarbodiimide,1.02g,5mmol) were added to dry dichloromethane,and the mixture was stirred at room temperature overnight to obtain reaction solution 3.The reaction solution 3 was washed with water three times,dried over anhydrous sodium sulfate,and distilled under reduced pressure to obtain a crude product.The crude product was separated and purified by gradient elution.The ratio range of the eluent petroleum ether-dichloromethane was 100:1~10:1(v / v),and a green solid was obtained,which was intermediate M3;

[0070] The HNMR spectrum information of intermediate M3 is as follows: 11H NMR (400 MHz, CDCl3) δ (ppm) 8.01 (d, J = 7.3 Hz, 1H), 7.83 - 7.40 (m, 3H), 7.23 - 7.05 (m, 3H), 7.03 (dd, J = 7.8, 1.5 Hz, 1H), 6.88 (dd, J = 7.6, 6.2 Hz, 3H), 6.81 - 6.69 (m, 2H), 6.54 (d, J = 8.9 Hz, 1H), 6.52 - 6.36 (m, 3H), 6.34 (dd, J = 8.9, 2.6 Hz, 1H), 3.95 (q, J = 7.0 Hz, 2H), 3.35 (q, J = 7.1 Hz, 4H), 1.47 (t, J = 7.0 Hz, 3H), 1.17 (t, J = 7.1 Hz, 6H).

[0071] The 13C NMR spectrum information of intermediate M3 is as follows: 13 13C NMR (101 MHz, CDCl3) δ (ppm) 169.58, 152.93, 152.81, 151.86, 149.58, 146.92, 146.87, 144.29, 143.75, 134.83, 129.56, 128.93, 127.36, 127.33, 125.78, 124.94, 124.08, 123.75, 122.77, 121.21, 119.42, 118.86, 116.93, 115.27, 115.24, 113.12, 108.43, 105.09, 103.06, 97.53, 84.14, 44.51, 42.18, 33.93, 12.77, 12.53.

[0072] Intermediate M3 (0.55 g, 1 mmol) and Lawesson's reagent (0.44 g, 1.08 mmol) were added to dry toluene, and the mixture was stirred at 90 °C for 4 h under a nitrogen atmosphere to obtain reaction solution 4. The reaction solution 4 was distilled under reduced pressure to obtain a crude product, and the crude product was separated and purified by gradient elution. The ratio range of the eluent petroleum ether - dichloromethane was 100:1 - 10:1 (v / v), and a yellow solid was obtained, which was the large Stokes shift fluorescent probe (fluorescent probe RHSBZ). Its structural formula is shown in Figure 1 ;

[0073] The 1H NMR spectrum information of fluorescent probe RHSBZ is as follows: 11H NMR (400 MHz, CDCl3) δ (ppm) 7.88 (d, J = 7.8 Hz, 1H), 7.56 (ddd, J = 8.8, 7.5, 1.3 Hz, 1H), 7.49 (dd, J = 10.8, 4.1 Hz, 1H), 7.14 (ddd, J = 9.3, 8.8, 4.5 Hz, 3H), 7.04 (dd, J = 7.9, 1.3 Hz, 1H), 6.88 (t, J = 7.4 Hz, 3H), 6.70 (d, J = 8.8 Hz, 2H), 6.66~6.52 (m, 3H), 6.34 (s, 3H), 3.94 (q, J = 6.9 Hz, 2H), 3.38~3.32 (m, 4H), 1.26 (s, 3H), 1.18 (d, J = 7.0 Hz, 6H) (as Figure 2 shown).

[0074] The 13C NMR spectral information of the fluorescent probe RHSBZ is as follows: 13 13C NMR (101 MHz, CDCl3) δ (ppm) 158.66, 157.19, 151.90, 150.93, 148.25, 145.86, 143.79, 142.33, 135.42, 134.52, 133.90, 129.88, 128.79, 128.55, 127.36, 127.31, 127.28, 126.78, 126.57, 124.01, 123.78, 122.91, 122.66, 119.01, 116.12, 115.18, 108.77, 108.74, 103.00, 102.91, 95.54, 44.50, 42.09, 29.71, 12.78, 12.55 (as Figure 3 shown).

[0075] The MS spectral information of the fluorescent probe RHSBZ: m / z 628.2087 [C 38 H 33 N3O2S2 + H] + (as Figure 4 shown).

[0076] Performance Test

[0077] I. UV absorption and fluorescence emission experiments of the fluorescent probe RHSBZ prepared in Example 1

[0078] The fluorescent probe RHSBZ prepared in Example 1 was added to the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system to make the concentration of the fluorescent probe RHSBZ 10 μM. Its ultraviolet-visible absorption light and fluorescence spectrum were measured. Then, HClO (20 μM) was added and measured again. The ultraviolet-visible absorption spectra of the fluorescent probe RHSBZ (10 μM) in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system with or without HClO (20 μM) before and after the response to HClO (20 μM) are shown in Figure 5 , and the fluorescence spectra before and after the response to HClO (20 μM) are shown in Figure 6 ( Figure 5 and Figure 6 In [the figures], RHSBZ represents before adding HClO, and RHSBZ-HClO represents after adding HClO. The same applies hereinafter). The normalized diagrams of the absorption and fluorescence spectra after the response to HClO (20 μM) are shown in Figure 7 .

[0079] The fluorescent probe RHSBZ (10 μM) prepared in Example 1 was added to the EtOH / PBS (1:1 v / v 5 mM, pH = 7.4) system. Then, 0 - 20 μM of HClO was added successively, and the measured ultraviolet-visible titration spectra are shown in Figure 8 , and the fluorescence emission titration spectra are shown in Figure 9 . The linear relationship diagram between the emission intensity of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 at 776 nm and the HClO concentration (0 - 5 μM) is shown in Figure 10 .

[0080] As Figures 5 - 10 shown, it can be seen that the emission wavelength of the fluorescent probe RHSBZ is in the near-infrared region, the Stokes shift reaches 148 nm, and the fluorescence intensity at the maximum emission peak shows a good linear relationship within the range of the HClO concentration (0 - 5 μM).

[0081] II. Selectivity of the fluorescent probe RHSBZ prepared in Example 1 for the fluorescence detection of HClO

[0082] The fluorescent probe RHSBZ prepared in Example 1 was separately dispersed in the EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system to prepare a test solution with a concentration of 1×10 -5 mol / L. Then, 2×10 -5 mol / L of different ROS / amino acids / metal ions (HClO, ONOO - , H2O2, ·OH, ROO·, t-BuOO·, 1 O2, t-BuOOH, NO2 - , GSH, Cys, Hcy, Fe2+ , Co 2+ , Ni 2 + , Cu 2+ , Zn 2+ ), after shaking well and standing still, test its ultraviolet absorption spectrum, and excite it at a wavelength of 610 nm, and test its fluorescence emission spectrum. The results are as Figures 11 - 12 shown. It can be seen that only HClO causes a significant increase in absorption intensity and a fluorescence emission peak, indicating that the probe RHSBZ has high selectivity for the detection of HClO.

[0083] III. Interference test of other ROS / amino acids / metal ions on the detection of HClO by the fluorescence probe RHSBZ

[0084] Take 16 5-ml EP tubes, and first mix the fluorescence probe RHSBZ prepared in Example 1 with a concentration of 1×10 -5 mol / L prepared in an EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system with HClO with a concentration of 2×10 -5 mol / L, and conduct fluorescence spectrum testing. After the testing is completed, add other ROS / amino acids / metal ions (ONOO - , H2O2, ·OH, ROO·, t-BuOO·, 1 O2, t-BuOOH, NO2 - , GSH, Cys, Hcy, Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ ) with the same concentration as HClO respectively. After mixing evenly, conduct fluorescence spectrum testing again under the same testing conditions. The results are as Figure 13 shown. From Figure 13 it can be seen that the fluorescence probe shows almost unchanged responses before and after the addition of ROS / amino acids / metal ions, indicating that the fluorescence probe RHSBZ has good anti-interference ability for the detection of HClO.

[0085] IV. Determination of the response time of the fluorescence probe RHSBZ to HClO

[0086] Prepare a stock solution of the fluorescence probe RHSBZ with a molar concentration of 10 μM in an EtOH / PBS (1:1 v / v, 5 mM, pH = 7.4) system, and add HClO with a molar concentration of 20 μM to it. After gently stirring, let HClO diffuse by itself and detect the change process of the fluorescence intensity at 776 nm over time. The results are as Figure 14As shown, fluorescence appeared in the probe within 5 s after the addition of HClO (20 μM), and the fluorescence intensity increased significantly with time. The fluorescence intensity reached its maximum at around 100 s and then remained stable, indicating that the probe RHSBZ would provide a rapid analytical method for the detection of HClO.

[0087] V. Influence of pH on the Detection of HClO by the Fluorescent Probe Obtained in Example 1

[0088] The pH of PBS was adjusted to 5 - 10 with 0.5 - 1.0 M NaOH / HCl solution, and then it was mixed with an equal volume of ethanol to obtain an EtOH / PBS (1:1 v / v, 5 mM) mixed solution system with different pH values. Then, the fluorescent probe RHSBZ prepared in Example 1 was added to it to prepare a fluorescent probe RHSBZ solution with a concentration of 10 μM at different pH values, and a scatter plot of the probe fluorescence intensity at 776 nm versus pH was obtained. Then, HClO with a molar concentration of 20 μM was added to measure the fluorescence intensity again. The results are as Figure 15 shown. The fluorescence intensity of the fluorescent probe RHSBZ prepared in the present invention remained basically stable in the range of pH = 5 - 10, and the fluorescence intensity after the addition of HClO remained basically stable in the range of pH = 6.5 - 8.5, indicating that the recognition of HClO by the fluorescent probe RHSBZ can be carried out under physiological pH conditions and has the potential to be applied to living cells and tissues.

[0089] VI. Cytotoxicity Test of the Fluorescent Probe RHSBZ on MCF-7 Cells

[0090] MCF-7 cells were selected and seeded in a 96-well cell culture plate. They were incubated in a 5% CO₂ incubator with 10% fetal bovine serum (FBS) and DMEM medium for 24 h. After they grew well, a negative control group (a cell group without RHSBZ, with an RHSBZ concentration of 0) was set. At the same time, the cells were incubated with 5 fluorescent probe RHSBZ solutions (0, 1.25, 2.5, 5, 10, 20 μM) prepared in Example 1 with concentration gradients for 24 h, and then incubated with 5 mg / mL MTT for another 4 h. After the incubation ended, the original medium was discarded, 150 μL DMSO was added, and the OD value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The results are as Figure 16 shown. The fluorescent probe RHSBZ was non-toxic to MCF-7 cells.

[0091] VII. Staining and Imaging Experiment of the Fluorescent Probe RHSBZ on Living Cells

[0092] The MCF-7 cells were cultured in a 37 °C, 5% CO2 incubator with 10% fetal bovine serum (FBS) and DMEM medium until completion. Four parallel groups were seeded in 35-mm laser confocal cell culture dishes and cultured for an additional 24 h. The first group (RHSBZ) was incubated with 10 μM of the fluorescent probe RHSBZ prepared in Example 1 for 30 min and immediately imaged and photographed under a laser confocal microscope. The second group (RHSBZ+LPS / PMA) pre-cultured the cells with 10 μM of the fluorescent probe RHSBZ prepared in Example 1 for 30 min, washed three times with PBS, and then incubated with LPS (1 μg / mL) / PMA (1 μg / mL) for 1 h, washed three times with PBS, and subjected to endogenous imaging and photographing under a confocal microscope. The third group (RHSBZ+LPS / PMA / NAC) pre-cultured the cells with 10 μM of the fluorescent probe RHSBZ prepared in Example 1, washed three times with PBS, incubated with LPS (1 μg / mL) / PMA (1 μg / mL) and NAC (3 mM) for 1 h, washed three times with PBS, and imaged and photographed under a confocal microscope. The fourth group (RHSBZ+HClO) was incubated with 10 μM of the fluorescent probe RHSBZ prepared in Example 1 for 30 min, washed three times with PBS, then added 20 μM HClO and incubated for 30 min, washed three times with PBS, and subjected to exogenous imaging and photographing under a confocal microscope. The imaging conditions for the four groups should be kept consistent. The results are as Figure 17 shown. In the figure, (a1), (b1), and (c1) represent the fluorescence staining imaging diagrams of the first group under Bright filed, Red filed, and Merged, respectively. (a2), (b2), and (c2) represent the fluorescence staining imaging diagrams of the second group under Bright filed, Red filed, and Merged, respectively. (a3), (b3), and (c3) represent the fluorescence staining imaging diagrams of the third group under Bright filed, Red filed, and Merged, respectively. (a4), (b4), and (c4) represent the fluorescence staining imaging diagrams of the fourth group under Bright filed, Red filed, and Merged, respectively. Figure 17 It shows that the fluorescent probe RHSBZ can detect exogenous and endogenous HClO in cells.

[0093] VIII. Fluorescent imaging experiment of the fluorescent probe RHSBZ on rat liver tissue sections

[0094] Prepare tissue sections from rat livers. Flatten one side of the tissue with a vibrating microtome, and take one of the tissue sections. Group 1: First, treat it with the fluorescent probe RHSBZ prepared in Example 1 at 10 μM, then place it with 20 μM HClO in an incubator at 37 °C for 60 min, wash it three times with PBS and then image. Group 2: Place the tissue section with the fluorescent probe RHSBZ prepared in Example 1 at 10 μM in an incubator at 37 °C for 60 min, wash it three times with PBS and then image. Collect the change of fluorescence intensity with the scanning depth in the z-scanning mode of a Zeiss LSM 880 confocal laser scanning microscope, and the excitation wavelength is 633 nm. The fluorescence imaging diagram of the fluorescent probe RHSBZ (10 μM) prepared in Example 1 in rat liver tissue is shown in Figure 18 , in the figure, (a) is the fluorescence imaging diagram of Group 1, and (b) is the fluorescence imaging diagram of Group 2; Figure 19 is the change diagram of fluorescence intensity with the scanning depth (both are pictures at 50 μm). From Figure 18 and 19 , it can be seen that in living tissues, the probe RHSBZ has good tissue penetration ability, with a penetration depth of up to 140 μm, and can realize the detection and imaging of HClO in tissues.

[0095] The above is only the preferred specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A large Stokes shift near-infrared fluorescent probe for detecting HClO, characterized in that, The molecular formula of the fluorescent probe is C 38 H 33 N3O2S2, and the structural formula is as follows:

2. A method for preparing the large Stokes shift near-infrared fluorescent probe according to claim 1, characterized in that, It includes the following steps: Using 2-methoxyphenothiazine as the starting material, dimethyl sulfoxide as the solvent, adding iodoethane under alkaline conditions, and heating and reacting under a protective atmosphere to obtain intermediate M1; Dissolving the intermediate M1 and 4-diethylaminoketonic acid in methanesulfonic acid, heating and reacting until complete, and adjusting the pH to neutral to obtain intermediate M2; Adding the intermediate M2, o-aminophenol, 4-dimethylaminopyridine and dicyclohexylcarbodiimide to dichloromethane and stirring overnight to obtain intermediate M3; Adding the intermediate M3 and Lawesson's reagent to toluene, and heating and stirring the reaction under a protective atmosphere to obtain the large Stokes shift near-infrared fluorescent probe; The structural formula of the intermediate M1 is The structural formula of the intermediate M2 is The structural formula of the intermediate M3 is 3. The preparation method of the large Stokes shift near-infrared fluorescent probe according to claim 2, wherein, The molar ratio of the 2-methoxyphenothiazine to the iodoethane is 1:2.

5.

4. The preparation method of the large Stokes shift near-infrared fluorescent probe according to claim 2, wherein, The molar ratio of the intermediate M1 to the 4-diethylaminoketonic acid is 1:

1.

5. The preparation method of the large Stokes shift near-infrared fluorescent probe according to claim 2, characterized in that, The molar ratio of the intermediate M2, o-aminophenol, 4-dimethylaminopyridine and dicyclohexylcarbodiimide is 1:1.1:1:2.

5.

6. The preparation method of the large Stokes shift near-infrared fluorescent probe according to claim 2, wherein, The molar ratio of the intermediate M3 to the Lawesson's reagent is 1:1.08.

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