Monothiophene Aza-BODIPY fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a monothiophene Aza-BODIPY fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes, and the structure of the fluorescent probe is shown as BOD (Biochemical Oxygen Demand)-5 in the specification. According to the invention, thiophene groups with very small molecular weight are introduced to 1, 7 sites of an Aza-BODIPY parent nucleus, so that absorption and emission wavelengths can be subjected to red shift to a near-infrared region, and meanwhile, the increase of molecular mass is not large. 4-hydroxyacetophenone is used as a raw material to introduce hydroxyl to serve as a reaction group of a signal molecule on the 3 and 5 sites of the Aza-BODIPY parent nucleus. 2, 4-dinitrobenzenesulfonyl chloride is selected as a recognition group and a fluorescence quenching group of the biological mercaptan. The fluorescent probe disclosed by the invention has accuracy and reliability on the detection of biological mercaptan, the application of the fluorescent probe in practice is greatly improved, and a new way is opened up for the analysis of biological mercaptan.

Description

Technical Field

[0001] The present application relates to the technical field of fluorescent probes, and in particular to a monothiophene Aza-BODIPY fluorescent probe and its preparation method and application. Background Art

[0002] Biothiols, such as cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), are widely present in cells and organisms and play a vital role in physiological and pathological processes. Abnormal levels of biothiols are associated with many diseases. For example, Cys deficiency is thought to be associated with growth retardation, liver damage, cardiovascular disease, and skin lesions. Hcy is a risk factor for heart disease and Alzheimer's disease. GSH, as the most abundant intracellular sulfhydryl group, plays an important role in endogenous antioxidant activity. High or low GSH levels may be related to AIDS, cancer, and many neurological diseases. Therefore, the detection and quantitative analysis of sulfhydryl groups in biological systems is of great significance, which may provide valuable information for the early diagnosis of certain diseases.

[0003] Fluorescent probes have become one of the most convenient methods in various detection technologies due to their simple operation, real-time monitoring, high sensitivity and non-invasiveness. To date, a large number of fluorescent thiol probes have been developed based on common fluorophores, such as blue-emitting coumarin, green-emitting fluorescein, green or yellow-emitting naphthalimide, and green or red-emitting BODIPY. However, these fluorescent probes have some problems: (1) The primary problem is the limitation of the photophysical properties of existing probes - their absorption spectra (λ abs ) and emission spectrum (λ em ) are mostly located in the visible light region of 400-650nm, resulting in significant endogenous fluorescence background interference and photon scattering effects in in vivo tissue imaging, resulting in decreased spatial resolution and deterioration of the signal-to-noise ratio; (2) Secondly, the recognition performance of the probe has defects such as insufficient sensitivity (the detection limit is generally higher than 10μM) and slow response kinetics greater than 30s, which makes it difficult to meet the needs of real-time dynamic monitoring; (3) Thirdly, the existing biothiol detection system is overly dependent on the single-mode output of fluorescence intensity and lacks multimodal coupling strategies such as mass spectrometry, ratiometric detection or photoacoustic imaging, which makes the analysis results susceptible to factors such as probe concentration, photobleaching and microenvironmental disturbances; (4) Finally, the development of molecular probes based on optical visualization analysis technology still has technical bottlenecks, and related research reports are still limited. Compared with traditional visible light region fluorescent dyes (λ em Near-infrared fluorescent probes (700-1700 nm) exhibit significant advantages in the field of biosensing due to their long wavelengths, which can effectively avoid interference from biological autofluorescence. Aza-BODIPY, a new near-infrared dye, is an ideal molecular framework for constructing high-performance near-infrared fluorescent probes.

[0004] Therefore, how to develop a monothiophene Aza-BODIPY fluorescent probe and its preparation method and its application in detecting biothiols are technical problems that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a monothiophene Aza-BODIPY fluorescent probe and a preparation method and application thereof.

[0006] A monothiophene Aza-BODIPY fluorescent probe, the structure of which is shown in formula BOD-5:

[0007]

[0008] The molecular formula of the fluorescent probe is C 40 H 22 BF2N7O 14 S4, molecular weight is 1001.0169.

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

[0010] (1) Synthesis of BOD-1:

[0011] Synthesis: 2-thiophenecarboxaldehyde and 4-hydroxyacetophenone were dissolved in anhydrous ethanol, and the mixture solution was cooled in a cold trap. KOH aqueous solution was added dropwise with stirring. After the solution was added dropwise, the reaction solution was transferred to a water bath and stirred for reaction. After the reaction was completed, the reaction solution was placed in a cold trap for cooling. HCl aqueous solution was added dropwise with stirring. After the addition was completed, a large amount of yellow solid precipitated. The mixture was filtered and the filter cake was washed with petroleum ether. The crude product was recrystallized to obtain a yellow solid, which was recorded as BOD-1.

[0012] (2) Synthesis of BOD-2:

[0013] BOD-1 and nitromethane were dissolved in methanol, and then anhydrous K2CO3 was added. The mixture was heated to reflux in an oil bath and stirred for reaction. After the reaction, the reaction solution was transferred to a cold trap for cooling, and an HCl aqueous solution was added dropwise. The solvent methanol was concentrated under reduced pressure. The crude product was dissolved in CH2Cl2. The organic phase was washed with brine several times and allowed to stand for separation. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove the solvent CH2Cl2. The crude product was then purified by silica gel chromatography to obtain a light yellow solid, which was recorded as BOD-2.

[0014] (3) Synthesis of BOD-3:

[0015] Under argon protection, ammonium acetate and BOD-2 were dissolved in ethanol, and the reaction solution was heated to reflux. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the ethanol solvent, and the crude product was dissolved in ethyl acetate. The organic phase was washed with water several times, allowed to stand for separation, and dried over anhydrous Na2SO4. Half of the solvent was concentrated under reduced pressure, and then transferred to a cold trap for cooling. N-hexane was added dropwise, and the mixture was filtered and the filter cake was washed with n-hexane to obtain a blue-black solid, recorded as BOD-3.

[0016] (4) Synthesis of BOD-4:

[0017] Under argon protection, BOD-3 was dissolved in anhydrous CH2Cl2, placed in a cold trap with stirring, and then N,N-diisopropylethylamine was added dropwise with stirring. Then, boron trifluoride etherate was added and the reaction was allowed to proceed at room temperature. After the reaction was completed, the mixture was washed with brine several times and separated. The organic phase was dried over anhydrous Na2SO4, and the solvent CH2Cl2 was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a red-black metallic solid, which was recorded as BOD-4.

[0018] (5) Synthesis of BOD-5:

[0019] Dissolve BOD-4 and triethylamine in anhydrous CH2Cl2, cool the reaction solution in a cold trap, add 2,4-dinitrobenzenesulfonyl chloride and react at room temperature. After the reaction is completed, remove the solvent CH2Cl2 by rotary evaporation, and then purify the crude product by column chromatography to obtain a blue-black solid, recorded as BOD-5.

[0020] Synthesis route:

[0021]

[0022] Furthermore, in step (1), the molar ratio of 2-thiophenecarboxaldehyde to 4-hydroxyacetophenone is 1.2:1, 1 mol of 4-hydroxyacetophenone is dissolved in 250 mL of anhydrous ethanol, and a KOH aqueous solution is added dropwise under stirring to adjust the system pH to 12-14, and the concentration of the KOH aqueous solution is 2.5 mol / L. An HCl aqueous solution is added dropwise under stirring to adjust the system pH to 2-3, and the concentration of the HCl aqueous solution is 2.5 mol / L;

[0023] The mixture solution was cooled to 0°C in a cold trap, and KOH aqueous solution was added dropwise with stirring. After the solution was added, the reaction solution was transferred to a water bath and stirred at 50°C for 24 hours. After the reaction was completed, the reaction solution was placed in a cold trap and cooled to 0°C.

[0024] Further, in step (2), the molar ratio of BOD-1, nitromethane and K2CO3 is 1:5:5, 1 mol of BOD-1 is dissolved in 250 mL of methanol, and the pH of the solution is adjusted to 2 by adding an aqueous HCl solution with a concentration of 2.5 mol / L, and the solvent methanol is removed by vacuum concentration, and 250 mL of CH2Cl2 is added to dissolve the crude product obtained from 1 mol of BOD-1;

[0025] The oil bath is heated to reflux, and the reaction is stirred for 24 h, and after the reaction is completed, the reaction solution is transferred to a cold trap and cooled to 0 DEG C.

[0026] Further, in step (3), the molar ratio of ammonium acetate and BOD-2 is 35:1, 1 mol of BOD-2 is dissolved in 150 mL of ethanol, and 250 mL of CH2Cl2 is added to dissolve the crude product obtained from 1 mol of BOD-1;

[0027] The reaction solution is heated to reflux for 48 h, half of the solvent is concentrated under reduced pressure, and then transferred to a cold trap and cooled to 0 DEG C.

[0028] Further, in step (4), the molar ratio of BOD-3, N,N-diisopropylethylamine and boron trifluoride ether is 1:30:40, 1 mol of BOD-3 is dissolved in 120 mL of anhydrous CH2Cl2;

[0029] BOD-3 is dissolved in anhydrous CH2Cl2 and placed in a cold trap at 0 DEG C and stirred for 15 min, then N,N-diisopropylethylamine is added dropwise and stirred for 15 min, then boron trifluoride ether is added, and the reaction is carried out at room temperature for 50 h.

[0030] Further, in step (5), the molar ratio of BOD-4, triethylamine and 2,4-dinitrobenzenesulfonyl chloride is 1:2:2, 1 mol of BOD-4 is dissolved in 20 mL of anhydrous CH2Cl2;

[0031] BOD-4 and triethylamine are dissolved in anhydrous CH2Cl2, the reaction solution is placed in a cold trap and cooled to 0 DEG C, 2,4-dinitrobenzenesulfonyl chloride is added, and the reaction is carried out at room temperature for 5 h.

[0032] The application also provides a use of the single thiophene Aza-BODIPY fluorescent probe in detecting biological thiols.

[0033] The application has the following beneficial effects:

[0034] The single thiophene Aza-BODIPY fluorescent probe provided by the application has the characteristics of high selectivity and high sensitivity when detecting biological thiols, can realize rapid detection of biological thiols, and has the advantages of simple operation in the detection process, and provides a new efficient and practical method for detecting biological thiols.

[0035] The present application introduces a thienyl group with very small molecular weight into the 1,7 position of an Aza-BODIPY mother nucleus, which can red shift the absorption and emission wavelength to the near-infrared region, while the increase in molecular weight is not large. In addition, 4-hydroxyacetophenone is used as a raw material to introduce a hydroxyl group as a signal molecule reaction group at the 3,5 position of the Aza-BODIPY mother nucleus. Finally, 2,4-dinitrobenzenesulfonyl chloride is selected as the recognition group and fluorescence quenching group of biological thiols. According to this design idea, the present application designs a near-infrared fluorescent probe based on Aza-BODIPY to detect biological thiols. The present application has accuracy and reliability in the detection of biological thiols, greatly improves its application in practice, and opens up a new way for the analysis of biological thiols. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a schematic diagram of the reaction mechanism of the fluorescent probe of the present application and biological thiols.

[0037] Figure 2 In the figure, a is the ultraviolet absorption spectrum of BOD-OH (30 μM), probe (5 μM) and after reaction with Cys, Hcy, GSH (30 μM); b is the fluorescence spectrum of BOD-OH (30 μM), probe (5 μM) and after reaction with Cys, Hcy, GSH (30 μM); c is the ultraviolet absorption fold line graph of the reaction of the probe with various amino acids; d is the ultraviolet absorption column chart of the reaction of the probe with various amino acids.

[0038] Figure 3 It is the H NMR graph of BOD-1. 1

[0039] Figure 4 It is the HRMS graph of BOD-1.

[0040] Figure 5 It is the H NMR graph of BOD-2. 1

[0041] Figure 6 It is the HRMS graph of BOD-2.

[0042] Figure 7 It is the H NMR graph of BOD-3. 1

[0043] Figure 8 It is the HRMS graph of BOD-3.

[0044] Figure 9 It is the H NMR graph of BOD-4. 1

[0045] Figure 10 It is the HRMS graph of BOD-4.​​​​

[0046] Figure 11 For BOD-5 1 H NMR spectrum.

[0047] Figure 12 This is the HRMS chart of BOD-5.

[0048] Figure 13 These are the UV absorption graphs of the probe reacting with different concentrations of biothiols, where Figure a is Cys, Figure b is Hcy, and Figure c is GSH; the UV absorption error scatter plots and linear relationship graphs of the probe reacting with different concentrations of biothiols, where Figures d and g are Cys, Figures e and h are Hcy, and Figures f and i are GSH.

[0049] Figure 14 Figures 2 and 3 are the fluorescence emission error scatter plots and linear relationship diagrams of the reaction between the probe and different concentrations of biological thiols, where Figures a and d are Cys, Figures b and e are Hcy, and Figures c and f are GSH.

[0050] Figure 15 Figure 3 is a temperature scatter plot of the probe after the reaction with different concentrations of biothiols in solution, where Figure a is Cys, Figure b is Hcy, and Figure c is GSH; Figure 4 is a temperature error point-line plot and linear relationship diagram of the probe after the reaction with different concentrations of biothiols in solution, where Figures d and g are Cys, Figures e and h are Hcy, and Figures f and i are GSH. DETAILED DESCRIPTION

[0051] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0052] Example 1

[0053] Synthesis of BOD-1:

[0054] Synthesis: In a 500 mL round-bottom flask, 2-thiophenecarboxaldehyde (10.75 g, 8.96 mL, 96 mmol, 1.2 eq.) and 4-hydroxyacetophenone (10.89 g, 80 mmol, 1 eq.) were dissolved in 250 mL of anhydrous ethanol. The mixture was then cooled to 0°C in a cold trap and aqueous KOH (70 mL, 2.5 M) was added dropwise with stirring. After the addition of the solution, the reaction solution was transferred to a water bath at 50°C and stirred for 24 h. The reaction progress was determined by TLC analysis.

[0055] Purification: After the reaction, the reaction solution was cooled to 0°C in a cold trap. Aqueous HCl (70 mL, 2.5 M) was added dropwise with stirring. A large amount of yellow solid precipitated after the addition. The mixture was filtered and the filter cake was washed with petroleum ether. The crude product was recrystallized to obtain a yellow solid (12.02 g, 65%). 1 HNMR(600MHz,Chloroform-d)δ8.01-7.96(m,2H),7.94(d,J=15.3Hz,1H),7.41(dt,J=5.0,1.0H z,1H),7.37-7.31(m,2H),7.09(dd,J=5.0,3.6Hz,1H),6.96-6.92(m,2H),6.07(d,J=3.4Hz,1H).

[0056] Synthesis of BOD-2:

[0057] In a 500 mL round-bottom flask, dissolve BOD-1 (12.02 g, 52 mmol, 1 eq.) and nitromethane (15.86 g, 13.95 mL, 260 mmol, 5 eq.) in 250 mL of methanol, then add anhydrous KCO (35.88 g, 260 mmol, 5 eq.). Heat to reflux in an oil bath and stir for 24 hours. TLC analysis determines the progress of the reaction. After completion, transfer the reaction solution to a cold trap, cool to 0°C, and add 2.5 M aqueous HCl dropwise to adjust the solution to pH 2. Concentrate under reduced pressure to remove the methanol solvent. Dissolve the crude product in 250 mL of CHCl. ​​Wash the organic phase three times with brine and allow to separate. Dry the organic phase over anhydrous NaSO and concentrate under reduced pressure to remove the CHCl solvent. The crude product was then purified by silica gel chromatography (petroleum ether / ethyl acetate (5 / 1, v / v)) to give a light yellow solid (4.72 g, 36.2%). 1 HNMR(600MHz,Chloroform-d)δ7.89-7.84(m,2H),7.20(dd,J=5.0,1.3Hz,1H),6.96-6.91(m,2H),6.88-6.84(m,2H),4 .84(dd,J=12.6,6.2Hz,1H),4.69(dd,J=12.6,7.7Hz,1H),4.57-4.49(m,1H),3.76(q,J=7.0Hz,1H),3.50-3.38(m,2H).

[0058] Synthesis of BOD-3:

[0059] Under argon, ammonium acetate (40.4 g, 525 mmol, 35 eq.) and BOD-2 (4.5 g, 15 mmol, 1 eq.) were dissolved in 150 mL of ethanol in a Schlenk vessel. The reaction solution was heated at reflux for 48 h. TLC analysis determined the progress of the reaction. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the ethanol solvent, and the crude product was dissolved in 150 mL of ethyl acetate. The organic phase was washed three times with water, allowed to stand, and dried over anhydrous Na2SO4. Half of the solvent was concentrated under reduced pressure, then cooled to 0°C, and n-hexane was added dropwise. The mixture was filtered, and the filter cake was washed with n-hexane to obtain a bluish-black solid (1 g, 13.3%). 1 HNMR(600MHz,DMSO-d6)δ7.92-7.88(m,6H),7.69(dd,J=5.1,1.1Hz,2H),7.42 (s,2H),7.21(dd,J=5.0,3.6Hz,2H),7.02-6.98(m,4H),4.22(t,J=6.6Hz,1H).

[0060] Synthesis of BOD-4:

[0061] Under argon, BOD-3 (0.95 g, 1.9 mmol, 1 eq.) was dissolved in 120 mL of anhydrous CH2Cl2 in a 250 mL Schlenk vessel. The mixture was placed in a cold trap (0°C) and stirred for 15 minutes. N,N-diisopropylethylamine (7.37 g, 9.42 mL, 57 mmol, 30 eq.) was then added dropwise and stirred for 15 minutes. Boron trifluoride etherate (10.71 g, 9.52 mL, 76 mmol, 40 eq.) was then added and allowed to react at room temperature for 50 hours. TLC analysis determined the progress of the reaction. After completion of the reaction, the mixture was washed three times with 60 mL of brine and separated. The organic phase was dried over anhydrous Na2SO4 and the CH2Cl2 solvent was removed by rotary evaporation. The crude product was purified by column chromatography (dichloromethane / methanol (100 / 1, v / v)) to yield a reddish-black metallic solid (0.31 g, 26.3%). 1 HNMR (600MHz, DMSO-d6) δ10.54(s,2H),8.04(dd,J=9.1,2.4Hz,6H),7.90(d,J=5.1Hz,2H),7.48(s,2H),7.29(dd,J=5.0,3.7Hz,2H),6.97-6.94(m,4H).

[0062] Synthesis of BOD-5:

[0063] In a 100 mL round-bottom flask, BOD-4 (100 mg, 0.18 mmol, 1 eq.) and triethylamine (36.3 mg, 0.05 mL, 0.36 mmol, 2 eq.) were dissolved in 20 mL of anhydrous CHCl. ​​The reaction solution was cooled to 0°C in a cold trap, and 2,4-dinitrobenzenesulfonyl chloride (96 mg, 0.36 mmol, 2 eq.) was added. The reaction was allowed to react at room temperature for 5 h, and the progress of the reaction was determined by TLC analysis. After the reaction was completed, the CHCl solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (petroleum ether / ethyl acetate (2 / 1, v / v)) to obtain a bluish-black solid (56 mg, 30.6%). 1 HNMR (600MHz, DMSO-d6) δ9.14(d,J=2.3Hz,2H),8.63(dd,J=8.7,2.3Hz,2H),8.36(d,J=8.7Hz,2H),8.09(s,7H),7.62-7.21(m,9H).

[0064]

[0065] Analysis of the reaction mechanism between fluorescent probes and biothiols

[0066] PET is a classic electron transfer process, which is often used to construct fluorescent probes. Typical PET fluorescent probes consist of three parts: fluorophore, linker and recognition group. Figure 1 The reaction mechanism of the probe is shown as follows: the Aza-BODIPY matrix acts as a fluorophore to transmit fluorescent signals, DNBS acts as a recognition and activation group, and nucleophilic aromatic substitution occurs between the probe and the thiol group of the biological thiol. The thiol group of the thiol attacks the carbon atom position of the benzene ring connected to the sulfonyl group in 2,4-dinitrobenzenesulfonyl chloride, causing the entire molecular structure to change, resulting in the interruption of PET and the onset of fluorescence.

[0067] UV absorption and fluorescence emission spectral analysis of fluorescent probes

[0068] In order to study the optical response of the fluorescent probe of the present invention to biothiols, the present invention studied the ultraviolet absorption spectrum and fluorescence emission spectrum of the fluorescent probe in DMSO / PBS (1:1, v:v; pH=7.4) solution. Figure 2 As shown in Figure a, the maximum absorption wavelength of the probe is at 730nm, which is located in the NIR band. Figure 2 As shown in Figure b, the presence of PET turns off the fluorescence of the probe, and the fluorescence emission is at 760nm with a fluorescence intensity of only about 250. When Cys, Hcy, and GSH are added, the probe is gradually converted into BOD-OH due to the cleavage of DNBS. Figure 2As can be seen from Figure a, the absorption wavelength gradually red-shifts from 730nm to 780nm, which is at the same wavelength as the maximum absorption of BOD-OH. This also preliminarily shows that the probe of the present invention successfully responds to biothiols Cys, Hcy and GSH. In addition, after adding Cys, Hcy and GSH, Figure 2 The fluorescence intensity in panel b increased several-fold to 1000 and 1500, respectively. This also confirmed the probe's reaction mechanism: upon binding to Cys, Hcy, and GSH, nucleophilic aromatic substitution by the thiol groups disrupted the PET reaction, leading to an increase in fluorescence intensity. These results demonstrate that our probe, based on the reactive properties of thiols, can successfully identify these three biothiols.

[0069] Probe selectivity analysis

[0070] Selectivity is another important parameter of fluorescent probes. To evaluate whether common amino acids in cells would interfere with the selectivity of the probe for biothiols, we tested the response of the probe (5 μM) to three biothiols and various amino acids (Cys, Hcy, GSH, Phe, Try, Lie, Leu, Ala, Pro, Glu, Thr, Gly, Ser, Asp) in DMSO / PBS (1:1, v:v; pH = 7.4) solution. Figure 2 Figures c and d show the UV absorption intensity of the test species (30 μM). The probe's native absorption peak is at 730 nm. The addition of Cys, Hcy, and GSH significantly enhances the probe's absorption intensity, and the absorption red-shifts due to structural changes triggered by nucleophilic aromatic substitution of the thiol group. However, in the presence of various amino acids, the change in absorption intensity is negligible, and the absorption peak at 730 nm remains essentially unchanged, indicating that the probe does not react with these amino acids. These results demonstrate the probe's good selectivity for thiols.

[0071] Photothermal testing of probe response to biothiols

[0072] 2 mL of the solution after the reaction between the probe and biothiol was taken into a centrifuge tube and irradiated under an 808 nm laser emitter for 10 minutes. The temperature change per minute at each concentration was recorded using the mobile phone software FLIR One.

[0073] UV absorption detection and analysis of different concentrations of biothiols by probe

[0074] In order to explore the concentration dependence of the probe, a concentration response experiment of the probe and biothiol was conducted. Figure 13As shown, the concentration dependence of the probe on Cys was first investigated in a DMSO / PBS (1:1, v:v; pH 7.4) buffer system. The probe exhibited a primary absorption peak at 730 nm, attributed to the coplanar structure of the BODIPY core. With the addition of Cys, the probe's absorption peak red-shifted, with absorption gradually increasing at 780 nm and a new absorption peak gradually decreasing in the 600-650 nm range. The probe's UV spectrum exhibited a colorimetric shift. Correspondingly, under sunlight, the solution color shifted from blue to pink-purple, enabling naked-eye observation of Cys. This color change is primarily due to the cleavage of the probe upon reaction with Cys, resulting in a structural change to BOD-OH and the interruption of the intermolecular PET. Under the same test conditions, the concentration dependence of the probe on Hcy and GSH was investigated, demonstrating similar properties to those of Cys.

[0075] Next, in order to further study the interaction between the probe and biothiols, 200 μL of the solution after the probe reacted with different concentrations of biothiols was transferred to a 96-well microplate, and 5 groups of parallel samples were placed in a microplate reader to test the absorbance change at 780 nm, and a UV error scatter plot of the reaction between the probe and different concentrations of biothiols was obtained. Figure 13 As shown, overall, the UV absorption of the three biothiols showed a trend of first increasing and then stabilizing in the range of 0-30μM. For Cys, the probe absorbance gradually increased in the range of 0-5μM, and gradually stabilized in the range of 5-30μM; for Hcy, the probe absorbance gradually increased in the range of 0-10μM, and gradually stabilized in the range of 10-30μM; for GSH, the probe absorbance gradually increased in the range of 0-5μM, and gradually stabilized in the range of 5-30μM. When Cys was in the concentration range of 0-5μM, there was a good linear relationship between the concentration and UV absorption, and the linear regression equation was: y = 0.02245x + 0.10268 (R 2 =0.99564); when Hcy is in the concentration range of 0-5 μM, the concentration and UV absorption have a good linear relationship, and the linear regression equation is: y=0.01303x+0.08883(R 2 =0.99203); when the concentration of GSH is in the range of 0-5 μM, the concentration and UV absorption have a good linear relationship, and the linear regression equation is: y=0.00861x+0.09922(R 2 =0.99261). The test results demonstrate that the probe can sensitively and quantitatively detect biothiols (Cys, Hcy, GSH) within a certain concentration range in solution.

[0076] Fluorescence detection analysis of different concentrations of biothiols by probes

[0077] Similarly, we will take 200 μL of the solution after the probe reacts with different concentrations of biological thiols and transfer it to a 96-well microplate, with 5 parallel samples for each concentration, and test the change in fluorescence intensity at 760 nm in the microplate reader to obtain the fluorescence error scatter plot of the probe reacting with different concentrations of biological thiols. As shown in Figure 14 Generally, the fluorescence intensity of the three biological thiols shows a trend of first increasing and then stabilizing in the range of 0-30 μM. For Cys, in the range of 0-5 μM, the probe fluorescence intensity gradually increases, and in the range of 5-30 μM, the probe fluorescence intensity gradually tends to saturation; for Hcy, in the range of 0-10 μM, the probe fluorescence intensity gradually increases, and in the range of 10-30 μM, the probe fluorescence intensity gradually tends to saturation; for GSH, in the range of 0-5 μM, the probe fluorescence intensity gradually increases, and in the range of 5-30 μM, the probe fluorescence intensity gradually tends to saturation. When Cys is in the concentration range of 0-5 μM, the fluorescence intensity has a good linear relationship with the concentration, and the linear regression equation is: y = 3994x + 2945 (R 2 = 0.99374); when Hcy is in the concentration range of 0-5 μM, the fluorescence intensity has a good linear relationship with the concentration, and the linear regression equation is: y = 2492x + 4557 (R 2 = 0.99203); when GSH is in the concentration range of 0-5 μM, the fluorescence intensity has a good linear relationship with the concentration, and the linear regression equation is: y = 1340x + 5799 (R 2 = 0.99449). The test results are basically consistent with the results of the above ultraviolet absorption detection.

[0078] Photothermal detection analysis of the probe for different concentrations of biological thiols

[0079] BODIPY molecules are widely used in photothermal therapy because of their excellent photothermal properties. Here, we want to detect biological thiols through the photothermal situation after the probe reacts with biological thiols, which is a relatively novel detection method. Therefore, first, take 200 μL of the solution after the probe reacts with Cys and transfer it to a centrifuge tube, irradiate it under an 808 nm wavelength exciter (power is 1 W), and record the temperature change from 1-10 min (use a mobile phone to connect an infrared thermal imager to detect temperature changes). As shown in Figure 15 Generally, the temperature of the system gradually increases with the concentration and the irradiation time, and then tends to be stable. For the same concentration, the temperature gradually increases with the irradiation time of the light source. At the same time, the temperature increases with the increase of the concentration. Under the same test conditions, the temperature changes of Hcy and GSH were studied, which have similar properties similar to Cys.

[0080] Next, in order to further study the photo-thermal changes of the probe and biological thiols, we recorded the temperature of the test, and subtracted the initial temperature T0 of the sample detection from the temperature of 0-30 μΜ range, 0-10 min per minute to obtain the temperature point line graph. As shown in Figure 15 Fig. 3, for Cys, in the range of 0-5 μΜ, the temperature of the probe gradually increased, in the range of 5-30 μΜ, the temperature increased slowly and then tended to be stable; for Hcy, in the range of 0-10 μΜ, the temperature gradually increased, in the range of 10-30 μΜ, the fluorescence intensity of the probe gradually tended to be stable; for GSH, in the range of 0-5 μΜ, the temperature gradually increased, in the range of 5-30 μΜ, the temperature gradually tended to be stable. When Cys was in the range of 0-5 μΜ, the temperature of different concentrations had a good linear relationship, the linear regression equation was: y = 2.19143x + 8.70467 (R 2 = 0.99589); when Hcy was in the range of 0-10 μΜ, the temperature of different concentrations had a good linear relationship, the linear regression equation was: y = 1.58909x + 8.87273 (R 2 = 0.99107); when GSH was in the range of 0-5 μΜ, the temperature of different concentrations had a good linear relationship, the linear regression equation was: y = 1.65714x + 8.55714 (R 2 = 0.99433). The test results prove that the probe can be used for sensitive and quantitative detection of biological thiols (Cys, Hcy, GSH) in a certain concentration range by photo-thermal method. This opens up a new way of thinking for subsequent researchers detecting biological thiols.

[0081] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A monothiophene Aza-BODIPY fluorescent probe, characterized in that: The structure of the fluorescent probe is shown in formula BOD-5:

2. A monothiophene Aza-BODIPY fluorescent probe according to claim 1, characterized in that: The molecular formula of the fluorescent probe is C 40 H 22 BF2N7O 14 S4, molecular weight is 1001.0169.

3. A method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 1 or 2, characterized in that: The steps include: (1) Synthesis of BOD-1: Synthesis: 2-thiophenecarboxaldehyde and 4-hydroxyacetophenone were dissolved in anhydrous ethanol, and the mixture solution was cooled in a cold trap. KOH aqueous solution was added dropwise with stirring. After the solution was added dropwise, the reaction solution was transferred to a water bath and stirred for reaction. After the reaction was completed, the reaction solution was placed in a cold trap for cooling. HCl aqueous solution was added dropwise with stirring. After the addition was completed, a large amount of yellow solid precipitated. The mixture was filtered and the filter cake was washed with petroleum ether. The crude product was recrystallized to obtain a yellow solid, which was recorded as BOD-1. (2) Synthesis of BOD-2: BOD-1 and nitromethane were dissolved in methanol, and then anhydrous K2CO3 was added. The mixture was heated to reflux in an oil bath and stirred for reaction. After the reaction, the reaction solution was transferred to a cold trap for cooling, and an HCl aqueous solution was added dropwise. The solvent methanol was concentrated under reduced pressure. The crude product was dissolved in CH2Cl2. The organic phase was washed with brine several times and allowed to stand for separation. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove the solvent CH2Cl2. The crude product was then purified by silica gel chromatography to obtain a light yellow solid, which was recorded as BOD-2. (3) Synthesis of BOD-3: Under argon protection, ammonium acetate and BOD-2 were dissolved in ethanol, and the reaction solution was heated to reflux. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the ethanol solvent, and the crude product was dissolved in ethyl acetate. The organic phase was washed with water several times, allowed to stand for separation, and dried over anhydrous Na2SO4. Half of the solvent was concentrated under reduced pressure, and then transferred to a cold trap for cooling. N-hexane was added dropwise, and the mixture was filtered and the filter cake was washed with n-hexane to obtain a blue-black solid, recorded as BOD-3. (4) Synthesis of BOD-4: Under argon protection, BOD-3 was dissolved in anhydrous CH2Cl2, placed in a cold trap with stirring, and then N,N-diisopropylethylamine was added dropwise with stirring. Then, boron trifluoride etherate was added and the reaction was allowed to proceed at room temperature. After the reaction was completed, the mixture was washed with brine several times and separated. The organic phase was dried over anhydrous Na2SO4, and the solvent CH2Cl2 was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a red-black metallic solid, which was recorded as BOD-4. (5) Synthesis of BOD-5: Dissolve BOD-4 and triethylamine in anhydrous CH2Cl2, cool the reaction solution in a cold trap, add 2,4-dinitrobenzenesulfonyl chloride and react at room temperature. After the reaction is completed, remove the solvent CH2Cl2 by rotary evaporation, and then purify the crude product by column chromatography to obtain a blue-black solid, recorded as BOD-5. Synthesis route:

4. The method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 3, characterized in that: In step (1), the molar ratio of 2-thiophenecarboxaldehyde to 4-hydroxyacetophenone is 1.2:1, 1 mol of 4-hydroxyacetophenone is dissolved in 250 mL of anhydrous ethanol, and a KOH aqueous solution is added dropwise under stirring to adjust the system pH to 12-14, and the concentration of the KOH aqueous solution is 2.5 mol / L. An HCl aqueous solution is added dropwise under stirring to adjust the system pH to 2-3, and the concentration of the HCl aqueous solution is 2.5 mol / L; The mixture solution was cooled to 0°C in a cold trap, and KOH aqueous solution was added dropwise with stirring. After the solution was added, the reaction solution was transferred to a water bath and stirred at 50°C for 24 hours. After the reaction was completed, the reaction solution was placed in a cold trap and cooled to 0°C.

5. The method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 3, characterized in that: In step (2), the molar ratio of BOD-1, nitromethane and K2CO3 is 1:5:5, each 1 mol of BOD-1 is dissolved in 250 mL of methanol, and an aqueous HCl solution is added dropwise to adjust the solution to pH = 2, the concentration of the aqueous HCl solution is 2.5 mol / L, and the solvent methanol is removed by concentration under reduced pressure. The crude product obtained by adding 250 mL of CH2Cl2 for each 1 mol of BOD-1 is dissolved; The mixture was heated to reflux in an oil bath and stirred for 24 h. After the reaction was completed, the reaction solution was transferred to a cold trap and cooled to 0°C.

6. The method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 3, characterized in that: In step (3), the molar ratio of ammonium acetate to BOD-2 is 35:1, 1 mol of BOD-2 is dissolved in 150 mL of ethanol, and 250 mL of CH2Cl2 is added to dissolve the crude product of 1 mol of BOD-1; The reaction solution was heated under reflux for 48 h, half of the solvent was concentrated under reduced pressure, and then transferred to a cold trap and cooled to 0°C.

7. The method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 3, characterized in that: In step (4), the molar ratio of BOD-3, N,N-diisopropylethylamine and boron trifluoride etherate is 1:30:40, and each 1 mol of BOD-3 is dissolved in 120 mL of anhydrous CH2Cl2; Dissolve BOD-3 in anhydrous CH2Cl2, place in a cold trap and stir at 0℃ for 15 minutes, then add N,N-diisopropylethylamine dropwise and stir for 15 minutes, then add boron trifluoride etherate and react at room temperature for 50 hours.

8. The method for preparing the monothiophene Aza-BODIPY fluorescent probe according to claim 3, characterized in that: In step (5), the molar ratio of BOD-4, triethylamine and 2,4-dinitrobenzenesulfonyl chloride is 1:2:2, and each 1 mol of BOD-4 is dissolved in 20 mL of anhydrous CH2Cl2; Dissolve BOD-4 and triethylamine in anhydrous CH2Cl2, cool the reaction solution to 0°C in a cold trap, add 2,4-dinitrobenzenesulfonyl chloride and react at room temperature for 5 hours.

9. Use of the monothiophene Aza-BODIPY fluorescent probe according to claim 1 or 2 in detecting biothiols.

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