CN116410175B - Hypochlorite near infrared fluorescent probe with large Stokes shift and preparation method and application thereof - Google Patents

Abstract

The invention discloses a hypochlorite near infrared fluorescent probe DCP-ClO with large Stokes displacement, and a preparation method and application thereof. The structural formula of DCP-ClO is shown in formula I. When excited at 655nm, DCP-ClO has a very weak fluorescence emission at 726 nm. When DCP-ClO and ClO ^‑ coexist, a new fluorescence emission peak appears at 755 nm. Along with the increase of ClO ^‑ concentration, the fluorescence intensity at 755nm is gradually enhanced, and the fluorescence emission intensity F _755nm at 755nm and the ClO ^‑ concentration have good linear relation, so that a system which has larger Stokes displacement (100 nm) and can detect ClO ^‑ and has excellent optical performance is formed, and the high-sensitivity and high-selectivity fluorescence detection of ClO ^‑ can be realized.

Description

A hypochlorite near-infrared fluorescent probe with large Stokes shift and its preparation method and application

Technical Field

The invention belongs to the technical field of analysis and detection, and in particular relates to a hypochlorite near-infrared fluorescent probe DCP-ClO with a large Stokes shift and a preparation method and application thereof.

Background Art

Reactive oxygen species (ROS) are important signaling molecules in life systems and are closely related to many physiological and pathological processes. Hypochlorite (ClO ^- ) is one of the most active species among all ROS. It is produced in vivo by myeloperoxidase (MPO) catalyzing hydrogen peroxide (H ₂ O ₂ ) and chloride ions (Cl ^- ) in phagocytic leukocytes and monocytes. ClO ^- plays an important role in the immune system, such as inhibiting inflammation, resisting pathogens, regulating cell apoptosis, and especially resisting bacterial invasion. However, endogenous ClO ^- is a double-edged sword for the body. Excessive ClO ^- produced in the body can cause abnormal changes in proteins, nucleic acids and lipids through oxidation/chlorination reactions, thereby leading to tissue damage and causing many immune system-related diseases, such as arthritis, kidney disease, cardiovascular disease, nervous system damage and even cancer. Therefore, detecting intracellular ClO ^- levels is of great significance for biological research and clinical diagnosis of many diseases.

At present, there are many traditional methods for detecting ^ClO- , such as iodine titration, potentiometric titration, coulometric titration and chromatography. These detection methods have high selectivity and sensitivity, but there are disadvantages such as cumbersome operation, high instrument price, and inability to detect at the cell and tissue level. Compared with these traditional methods, the fluorescence detection method based on fluorescent probe technology has the characteristics of simple operation, low cost, real-time monitoring in cells or tissues, and strong anti-interference ability. However, the small molecule fluorescent probes currently used to detect ^ClO- generally have the disadvantages of short emission wavelength and being easily interfered by biological background fluorescence. Near-infrared fluorescent probes can effectively reduce the interference of background fluorescence and improve the sensitivity of detection.

Therefore, the development of a ClO ^- near-infrared fluorescent probe with fast response, large emission wavelength and Stokes shift is of great significance for the real-time monitoring of ^ClO- in biological cells and tissues.

Summary of the invention

An object of the present invention is to provide a near-infrared fluorescent probe molecule, DCP-ClO, which has a large Stokes shift characteristic and can quickly identify ClO ^- .

The DCP-ClO provided by the present invention has a structural formula as shown in Formula I:

Another object of the present invention is to provide a method for preparing DCP-ClO shown in Formula I.

The preparation method of DCP-ClO provided by the present invention comprises the following steps (see FIG1 for the preparation flow chart):

1) Synthesizing dicyanoisophorone (Compound 1) using isophorone and malononitrile as raw materials;

2) Using cyclopentanone as a raw material, compound 2 is generated under the action of phosphorus tribromide (PBr ₃ );

3) Compound 2 reacts with 2-hydroxy-4-methoxybenzaldehyde to generate compound 3;

4) Compound 3 is demethylated under the action of boron tribromide (BBr ₃ ) to generate compound 4;

5) Using compound 1 and compound 4 as raw materials to synthesize compound DCP-OH;

6) reacting the compound DCP-OH with dimethylthiocarbamoyl chloride to obtain the compound DCP-ClO represented by formula I;

In step 1) of the above method, the specific method for synthesizing dicyanoisophorone (compound 1) using isophorone and malononitrile as raw materials is: isophorone, malononitrile, piperidine and acetic acid are reacted under nitrogen protection with ethanol as solvent to obtain compound 1; the reaction temperature of the reaction is 90°C and the reaction time is 6h; in the reaction, the molar ratio of isophorone, malononitrile, piperidine and acetic acid is 1:1.1:0.09:0.09.

In step 2) of the above method, the specific method of using cyclopentanone as a raw material to generate compound 2 under the action of phosphorus tribromide (PBr ₃ ) is as follows: phosphorus tribromide (PBr ₃ ) is added to a mixed solution of DMF and CHCl ₃ at 0°C, stirred for 45 minutes, and then cyclopentanone is added, and then stirred and reacted at room temperature (25°C) for 16 hours to obtain compound 2; the molar ratio of cyclopentanone to phosphorus tribromide (PBr ₃ ) in the reaction is 1:2.3; and the volume ratio of DMF to CHCl ₃ in the mixed solution is 1:4.5.

In step 3) of the above method, the specific method for reacting compound 2 and 2-hydroxy-4-methoxybenzaldehyde to generate compound 3 is: reacting compound 2, 2-hydroxy-4-methoxybenzaldehyde and cesium carbonate in DMF to obtain compound 3; in the reaction, the molar ratio of compound 2, 2-hydroxy-4-methoxybenzaldehyde and cesium carbonate is 1:1.2:2.5; the reaction is carried out under stirring conditions, the reaction temperature of the reaction is 25°C, and the reaction time is 16h.

In step 4) of the above method, the specific method of removing the methyl group of compound 3 under the action of boron tribromide (BBr ₃ ) to generate compound 4 is as follows: dissolving compound 3 in anhydrous dichloromethane, slowly adding boron tribromide (BBr ₃ ) under ice bath, stirring at 0° C. for 1 hour, then slowly warming to room temperature (25° C.), and continuing the reaction for 4 hours to obtain compound 4. The above reaction is carried out under nitrogen protection, and the molar ratio of compound 3 to boron tribromide (BBr ₃ ) is 1:30.

In step 5) of the above method, the specific method for synthesizing DCP-OH using compound 1 and compound 4 as raw materials is: dissolving compound 1 and compound 4 in acetonitrile, then adding piperidine and acetic acid in sequence, and reacting under nitrogen protection to obtain compound DCP-OH; in the reaction, the molar ratio of compound 1, compound 4, piperidine and acetic acid is 1:1:5:8.7; the reaction temperature of the reaction is 80°C, and the reaction time is 14h.

In step 6) of the above method, the specific method of reacting the compound DCP-OH and dimethylthiocarbamoyl chloride to obtain the compound DCP-ClO shown in formula I is: dissolving the compound DCP-OH, dimethylthiocarbamoyl chloride and N,N-diisopropylethylamine (DIPEA) in anhydrous dichloromethane, and reacting under nitrogen protection to obtain the compound DCP-ClO; in the reaction, the molar ratio of the compound DCP-OH, dimethylthiocarbamoyl chloride and DIPEA is 1:12:4.3; the reaction temperature of the reaction is 25°C, and the reaction time is 4h.

Another object of the present invention is to provide the use of DCP-ClO.

The DCP-ClO provided by the present invention is used for at least one of the following 1)-7):

1) Fluorescent probe made of DCP-ClO;

2) Application of DCP-ClO as a fluorescent probe or as a fluorescent probe for detecting ClO ^- ;

3) Chemical sensor containing DCP-ClO;

4) Application of DCP-ClO in the preparation of chemical sensors or chemical sensors for detecting ^ClO- ;

5) Application of DCP-ClO in the detection of ClO ^- ;

6) Application of the fluorescent probe described in 1) above in detecting ClO ^- ;

7) Application of the chemical sensor described in 3) above in detecting ClO ^- .

The fluorescent probe or chemical sensor may be applied to a water body, such as an actual water sample, including drinking water, tap water, and the like.

The inventors of the present invention have experimentally confirmed that DCP-ClO itself has a weaker fluorescence emission at 726nm. When DCP-ClO coexists with ^ClO- , a new fluorescence emission peak appears at 755nm in the system. As the concentration of ^ClO- increases, the fluorescence at 755nm gradually increases, and _the fluorescence emission intensity _F755nm at 755nm has a good linear relationship with the concentration of ^ClO- , and has a high sensitivity to the detection of ^ClO- . Therefore, DCP-ClO is suitable for high-sensitivity detection of ^ClO- , which can be performed by fluorescence spectroscopy.

When fluorescence spectroscopy was used to detect ClO ^- with DCP-ClO as the detection reagent, an immediate response could be achieved, and the detection limit of the method was 3.95×10 ^-8 M, indicating that the sensor molecule DCP-ClO had a good response speed and sensitivity to ClO ^- , which was better than the sensitivity of similar near-infrared fluorescent probes reported so far for detecting ClO ^- . At the same time, DCP-ClO has good selectivity for the fluorescence response of ^ClO- , and DCP-ClO has ^no response to common ions and interfering substances (such as K ⁺ , Ca2 ⁺ , Zn2 ⁺ , Na ⁺ , Cu2 ^{+, Mg2+ , Fe2 +} , Fe3 ⁺ , ^Cl- , _NO3- , ^NO2- , _SO32- , ^HSO3- , _SO42- ^, _S2- , _GSH , Hcy, Cys, _H2O2 , OH, TBHP, OtBu, ^{1O2 ,} ON and _ONOO- ^, etc.) ^, and has high _detection specificity. In addition, when DCP-ClO is used to detect ^ClO- , due to its high detection sensitivity, only a small amount of sample is needed to complete the detection, which broadens the application range of this method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the preparation of DCP-ClO.

FIG2 is the H NMR spectrum of compound 1.

FIG3 is the carbon NMR spectrum of compound 1.

FIG4 is a high-resolution mass spectrum of compound 1.

FIG5 is the H NMR spectrum of compound 3.

FIG6 is the carbon NMR spectrum of compound 3.

FIG7 is a high-resolution mass spectrum of compound 3.

FIG8 is the H NMR spectrum of compound 4.

FIG9 is the carbon NMR spectrum of compound 4.

FIG10 is a high-resolution mass spectrum of compound 4.

FIG11 is a hydrogen nuclear magnetic resonance spectrum of DCP-OH.

FIG12 is the carbon NMR spectrum of DCP-OH.

FIG13 is a high-resolution mass spectrum of DCP-OH.

FIG. 14 is a hydrogen nuclear magnetic resonance spectrum of DCP-ClO.

FIG. 15 is the carbon NMR spectrum of DCP-ClO.

FIG16 is a high-resolution mass spectrum of DCP-ClO.

Figure 17 is a graph showing the ultraviolet absorption spectra of the system before and after the reaction of DCP-ClO (10 μM) and ClO ^- (140 μM) and DCP-OH (10 μM) (a); a graph showing the fluorescence spectra of the system before and after the reaction of DCP-ClO (10 μM) and ClO ^- (140 μM) and DCP-OH (10 μM) (b); a graph showing the change in the ultraviolet absorption spectrum of the coexistence system of DCP-ClO (10 μM) and different concentrations of ClO ^- (0-180 μM) (c); a graph showing the change in the fluorescence spectrum of the coexistence system of DCP-ClO (10 μM) and different concentrations of ClO ^- (0-180 μM) (d); a graph showing the relationship between the fluorescence intensity of the system and the concentration of ClO ^{- in the coexistence system of DCP-ClO (10 μM) and different concentrations of ClO -} (0-180 μM) (e); a graph showing the relationship between the fluorescence intensity of the system and the concentration of ClO - in the coexistence system of DCP-ClO (10 μM) and different concentrations of ClO ^- (f) The linear relationship between the fluorescence intensity of the system and the concentration of ClO- in the coexistence system (0-100μM).

Figure 18 shows the change of fluorescence intensity of DCP-ClO (10 μM) to ClO ^- (140 μM) over time (a); the change of fluorescence intensity of the coexistence system of DCP-ClO (10 μM) and ClO ^- (140 μM) and the system of DCP-ClO (10 μM) alone at different pH values (b); the selectivity of DCP-ClO (10 μM) to various ions, amino acids, reactive oxygen and reactive nitrogen (c); the anti-interference of DCP-ClO (10 μM) to various reactive oxygen and reactive nitrogen (d).

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

Example 1. Preparation of chemical sensor molecule DCP-ClO

The reaction process is shown in Figure 1, and the specific method is as follows:

Dissolve malononitrile (661 mg, 10 mmol) in ethanol (20 mL), add isophorone (1344 μL, 9 mmol), piperidine (79 μL, 0.8 mmol) and acetic acid (46 μL, 0.8 mmol). Under nitrogen protection, reflux at 90 ° C and stir for 6 hours. After the reaction is completed, cool to room temperature (25 ° C), slowly pour the solution into distilled water, precipitate solids, and filter. Dissolve the solid in ethanol and recrystallize to obtain gray solid compound 1 with a yield of 60.7%.

PBr ₃ (6.13 mL, 65.25 mmol) was added to a mixed solution of DMF (5.6 mL, 72.5 mmol) and chloroform (25 mL) at 0°C. After 45 min, cyclopentanone (2.5 mL, 28.25 mmol) was added and stirred at room temperature (25°C) for 16 h. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate (NaHCO ₃ ) solution. The mixture was extracted with dichloromethane and washed with a saturated sodium chloride (NaCl) solution. The organic phase was collected and dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), the solid was removed by filtration, and the solvent was removed by rotary evaporation to obtain a yellow-brown oily compound 2 with a yield of 70.4%. The substance was used directly in the next step without purification.

Compound 2 (2.6 g, 15 mmol), 2-hydroxy-4-methoxybenzaldehyde (2.739 g, 18 mmol) and cesium carbonate (12.2 g, 37.5 mmol) were added to DMF (18.75 mL) and stirred at room temperature (25 ° C) for 16 h. After the reaction was completed, the cesium carbonate residue was filtered off. The filtrate was concentrated, dissolved in ethyl acetate, washed with saturated sodium chloride (NaCl) solution, and extracted with ethyl acetate. The organic phase was collected and dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), the solid was filtered off, and the solvent was evaporated by rotary evaporation. Purification was performed by column chromatography, and the eluent was petroleum ether (boiling range 60-90 ° C)/ethyl acetate (15:1, v/v), and a yellow-brown solid compound 3 was obtained with a yield of 42%.

Compound 3 (228 mg, 1 mmol) was dissolved in anhydrous dichloromethane (30 mL) and placed in an ice bath under nitrogen protection. BBr ₃ (2.89 mL, 30 mmol) was slowly added, and after stirring for 1 h in an ice bath, the temperature was slowly raised to room temperature (25°C) and the reaction was continued for 4 h. After the reaction was completed, a saturated sodium bicarbonate (NaHCO ₃ ) solution was added in an ice bath to quench the reaction and adjust the pH to neutral. The mixture was washed with a saturated sodium chloride (NaCl) solution and extracted with dichloromethane/methanol (10:1, v/v). The organic phase was collected, dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), the solid was filtered off, and the solvent was evaporated by rotary evaporation. The mixture was purified by column chromatography, and the eluent was petroleum ether (boiling range 60-90°C)/ethyl acetate (1:1, v/v) to obtain a yellow solid compound 4 with a yield of 94.3%.

Compound 4 (214.2 mL, 1 mmol) and compound 1 (186.3 mg, 1 mmol) were dissolved in acetonitrile (40 mL), and piperidine (0.5 mL, 5 mmol) and acetic acid (0.5 mL, 8.7 mmol) were added in sequence. Under nitrogen protection, the mixture was refluxed at 80 ° C and stirred for 14 h. After the reaction, the solvent was removed by rotary evaporation and purified by column chromatography. The eluent was petroleum ether (boiling range 60-90 ° C)/ethyl acetate (5:1, v/v), and a black solid compound DCP-OH was obtained with a yield of 53.3%.

Dissolve DCP-OH (76.5 mg, 0.2 mmol) and N, N-dimethylthiocarbamoyl chloride (296.6 mg, 2.4 mmol) in anhydrous dichloromethane (2 mL), and slowly add DIPEA (150 μL, 0.86 mmol). Stir at room temperature (25 ° C) for 4 h under nitrogen protection. After the reaction is completed, remove the solvent and purify by column chromatography. The eluent is dichloromethane/petroleum ether (boiling range 60-90 ° C) (1:1, v/v) to obtain a black solid compound DCP-ClO with a yield of 20%.

The results of NMR and high resolution identification of compound 1: ¹ H NMR (CDCl ₃ , 500MHz) δ＝6.61(s, 1H); 2.50(s, 2H); 2.17(s, 2H); 2.02(s, 3H); 1.00(s, 6H). ¹³ C NMR (CDCl ₃ , 125MHz) δ＝170.5, 159.9, 120.7, 113.3, 112.5, 78.3, 45.8, 42.7, 32.5, 27.9(2C), 25.4. The H NMR spectrum and C NMR spectrum are shown in Figures 2 and 3 respectively. Instrument model: Bruker Avance 500MHz spectrometer. High-resolution mass spectrometry identification results of compound 1: HR-MS (ESI, m/z) cacld for C ₁₂ H ₁₅ N ₂ ⁺ [M+H] ⁺ :187.1235, found 187.1227. The results are shown in Figure 4. Instrument model: UPLC-Q/TOF Xevo G2-XS. The above results show that the obtained compound is indeed the target compound 1.

The results of NMR and high resolution identification of compound 3: ¹ H NMR (CD ₂ Cl ₂ , 500MHz) δ＝9.96(s,1H);7.05(d,J＝8.4Hz,1H);6.68-6.66(m,2H);6.56(s,1H);3.80(s,3H);2.71-2.69(m,2H);2.63-2.61(m,2H). ¹³ C NMR (CD ₂ Cl ₂ ,150MHz)δ＝183.8,163.7,161.0,152.9,137.5,127.5,121.9,116.4,115.6,110.9,101.5,55.7,24.3,23.6. The H NMR spectrum and C NMR spectrum are shown in Figures 5 and 6 respectively. Instrument model: Bruker Avance 500MHz spectrometer. High-resolution mass spectrometry identification results of compound 3: HR-MS (ESI, m/z) cacld for C ₁₄ H ₁₂ O ₃ ⁺ [M+H] ⁺ :229.0865, found 229.0866. The results are shown in Figure 7. Instrument model: UPLC-Q/TOF Xevo G2-XS. The above results show that the obtained compound is indeed the target compound 3.

NMR and high-resolution identification results of compound 4: ¹ H NMR (CDCl ₃ , 800 MHz) δ＝9.88(s, 1H), 7.16(d, J＝8.1Hz, 1H); 6.82(s, 1H); 6.63(s, 1H), 6.62(dd, J ₁ ＝8.7Hz, J ₂ ＝2.2Hz, 1H); 2.69 (t, J ＝ 6.2 Hz, 2H); 2.53 (d, J ＝ 6.0 Hz, 2H). ¹³ C NMR (CDCl ₃ , 200 MHz) δ =182.7,163.4,159.3,152.2,135.4,128.0,122.9,115.1,113.4,114.4,102.6,23.6,23.3. The H NMR spectrum and C NMR spectrum are shown in Figures 8 and 9, respectively. Instrument model: Bruker Avance III-800MHz spectrometer. High-resolution mass spectrometry identification results of compound 4: HR-MS (ESI, m/z) cacld for C ₁₃ H ₁₀ O ₃ ⁺ [M+H] ⁺ :215.0708, found 215.0708. The results are shown in Figure 10. Instrument model: UPLC-Q/TOF Xevo G2-XS. The above results show that the obtained compound is indeed the target compound 4.

The results of nuclear magnetic resonance and high-resolution identification of DCP-OH: ¹ H NMR (CDCl ₃ , 800MHz) δ＝10.01(s,1H),7.26(d,J＝15.3Hz,1H);7.05(d,J＝8.2Hz,1H);6.68(s,1H);6.56(dd,J ₁ ＝7.6Hz,J ₂ ＝2.2Hz,2H);6.53(dd,J ₁ ＝8.2Hz,J ₂ ＝2.2Hz,1H);6.43(d,J＝15.3Hz,1H);3.172(s,2H);3.165(s,2H);2.71(t,J＝4.8Hz,2H);2.659(t,J＝5.9Hz,2H);1.01(s,6H). ¹³ C NMR (CDCl ₃ , 200 MHz) δ＝169.1, 158.6, 156.7, 155.8, 152.9, 136.8, 129.4, 127.4, 126.4, 120.5, 118.8, 116.5, 114.8, 114.7, 113.9, 111.8, 102.6, 72.2, 48.6, 42.3, 38.1, 31.6, 27.4, 25.4, 23.8. The H NMR spectrum and the C NMR spectrum are shown in Figures 11 and 12 respectively. Instrument model: Bruker Avance III-800 MHz spectrometer. High resolution mass spectrometry identification results of DCP-OH: HR-MS (ESI, m/z) cacld for C ₂₅ H ₂₂ N ₂ O ₂ ⁺ [M] ⁺ :382.1681, found 382.1683. The results are shown in Figure 13. The above results show that the obtained compound is indeed the target compound DCP-OH.

The results of nuclear magnetic resonance and high resolution identification of DCP-ClO: ¹ H NMR (CDCl ₃ , 500MHz) δ＝7.16 (d, J＝15.6Hz, 1H); 7.01 (d, J＝8.3Hz, 1H); 6.82 (d, J＝1.8Hz, 1H); 6.73 (dd, J ₁ ＝8.2Hz, J ₂ =2.5Hz, 1H); 6.70 (s, 1H); 6.31 (s, 1H); 6.22 (d, J = 15.5Hz, 1H); 3.45 (s, 3H); 3.34 (s, 3H); 2.73 (d, J = 6.2Hz, 2H); 2.67 (d, J = 6.4Hz, 2H); 2.55 (s, 2H); 2. 42(s,2H); 1.06(s,6H). ¹³ C NMR (CDCl ₃ ,125MHz)δ＝187.4,168.8,155.4,155.1,153.9,152.7,140.9,129.0,127.0,126.3,121.9,121.4,118.5,117.5,117.3,114.5,113.7,111.0,75.5,43.5,43.1,39.3,38.9,32.1,28.1(2C),25.9,24.7. The H NMR spectrum and C NMR spectrum are shown in Figures 14 and 15 respectively. Instrument model: Bruker Avance 500MHz spectrometer. High-resolution mass spectrometry identification results of DCP-ClO: HR-MS (ESI, m/z) cacld for C ₂₈ H ₂₇ N ₃ O ₂ S ⁺ [M+Na] ⁺ :492.1722, found 492.1718. The results are shown in Figure 16. Instrument model: UPLC-Q/TOF XevoG2-XS. The above results show that the obtained compound is indeed the target compound DCP-ClO.

Example 2: DCP-ClO as an analytical reagent for fluorescence detection of ^ClO

1. Sensitivity of fluorescence detection of ^ClO- by DCP-OH

In a 5 mL plastic EP tube, 40 μL of DCP-ClO stock solution (1.0 mM, solvent: DMSO) and a certain volume of phosphate buffer solution (PBS)/acetonitrile mixed solution (1:1, v/v, PBS concentration: 10 mM, pH = 7.4) were added, and then different volumes of 10 mM ClO ^{-solution were added respectively, and then the volume was fixed to 4 mL with phosphate buffer solution (PBS)/acetonitrile mixed solution (1:1, v/v, PBS concentration: 10 mM, pH = 7.4), so that the final concentrations of ClO- in} each sample tube were 0, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160 and 180 μM, respectively, and the final concentration of DCP-ClO was 10 μM. After shaking each sample tube evenly, transfer it to a 1 cm quartz cell, and measure the ultraviolet absorption spectrum and fluorescence spectrum of the reaction system. Figure 17 (a) shows the ultraviolet absorption spectrum of the system before and after the reaction of DCP-ClO (10 μM) and ClO ^- (140 μM) and DCP-OH (10 μM). It can be seen from the figure that the maximum absorption wavelength of DCP-ClO is at 544 nm. After adding ClO ^- , the ultraviolet absorption wavelength of the system red-shifts to 570 nm, and basically coincides with the ultraviolet absorption spectrum of the fluorescent matrix DCP-OH. Figure 17 (b) shows the fluorescence spectrum of the system before and after the reaction of DCP-ClO (10 μM) and ClO ^- (140 μM) and DCP-OH (10 μM), with an excitation wavelength of 655 nm. It can be seen from the figure that DCP-ClO has a weaker fluorescence emission at 726 nm. After adding ClO ^- , the emission wavelength red-shifted to 755nm, the fluorescence intensity was significantly enhanced, and basically coincided with the fluorescence spectrum of the fluorescent matrix DCP-OH; Figure 17 (c) and (d) are the ultraviolet absorption spectrum change diagram and fluorescence spectrum change diagram of the coexistence system of DCP-ClO (10μM) and different concentrations of ClO ^- (0-180μM), respectively. It can be seen that with the increase of ClO ^- concentration, the maximum emission wavelength and ultraviolet absorption wavelength of the system gradually red-shifted, the fluorescence intensity gradually increased, and the ultraviolet maximum absorbance gradually decreased.

Figures 17(e) and (f) show the relationship between the fluorescence intensity of the system and the concentration and the linear relationship in the coexistence system of DCP-ClO (10μM) and ClO ^- (0-180μM). As can be seen from Figure (e), as the concentration of ClO ^- increases, the fluorescence intensity of the system (F _{755 nm} ) gradually increases. When the concentration of ClO ^- is greater than 100μM, the fluorescence intensity of the system no longer changes significantly. As can be seen from Figure 17(f), the fluorescence intensity of the system has a good linear relationship with the concentration of ClO ^- between 0 and 100μM.

FIG18(a) is a graph showing the fluorescence intensity of DCP-ClO (10 μM) versus ClO ^- (140 μM) over time. It can be seen from the figure that after adding ClO ^- , the fluorescence intensity of the system increases to the maximum value almost instantly and no longer changes significantly, indicating that DCP-ClO responds very quickly to ClO ^- and can achieve an immediate response effect. FIG18(b) is a graph showing the fluorescence intensity changes of the coexistence system of DCP-ClO (10 μM) and ClO ^- (140 μM) and the system when DCP-ClO (10 μM) exists alone at different pH values. It can be seen from the figure that when the pH is in the range of 4.0 to 10.0, the fluorescence intensity of the system with only DCP-ClO has almost no change and the value is low; when DCP-ClO and ClO ^- exist at the same time, in the pH range of 4.0 to 6.0, the fluorescence intensity of the system changes slightly, but the amplitude is small, indicating that DCP-ClO does not respond significantly to ClO ^- under acidic conditions. At pH 7, the fluorescence intensity of the system was significantly enhanced, and reached the maximum at pH = 7.4, indicating that DCP-ClO can effectively recognize ClO ^- under physiological conditions. When the pH was in the range of 8 to 10, the fluorescence intensity of the system began to decrease but was still higher than that under acidic conditions, indicating that DCP-ClO can also respond to ClO ^- under weakly alkaline conditions.

The above results show that the analytical reagent—DCP-ClO has excellent performance and can achieve high sensitivity and rapid fluorescence detection of ClO ^- .

2. Selectivity of DCP-ClO for fluorescence detection of ClO ^- .

At the same time, take several 5mL EP tubes and perform similar operations as above, replacing the added ^ClO- with various ions, amino acids, reactive oxygen and reactive nitrogen species, respectively. No. 1 to 27 correspond to: Blank, K ⁺ , Ca ²⁺ , Zn ²⁺ , Na ⁺ , Cu ²⁺ , Mg ²⁺ , Fe ^{2 +} , Fe ³⁺ , Cl ^- , NO ₃ ^- , NO ₂ ^- , SO ₃ ^2- , HSO ₃ ^- , SO ₄ ^2- , S ^2- , GSH, Hcy, Cys, H ₂ O ₂ , ·OH, TBHP, ·OtBu, ¹ O ₂ , ·ON, ONOO ^- and ClO ^- , among which the concentrations of No. 2 to 26 are 1mM, the concentration of No. 27 is 140μM, and No. 1 is the blank control group. The test results are shown in Figure 18(c). It can be seen from Figure 18(c) that the fluorescence intensity (F _{755 nm} ) of the system of sample tubes 2 to 26 after adding the above substances did not change significantly compared with the blank control group of sample tube 1, while the fluorescence intensity (F _{755 nm} ) of the system of sample tube 27 after adding ClO ^- increased significantly. The above phenomenon shows that DCP-ClO has no response to other substances except ClO ^- , so as a detection reagent, DCP-ClO has good selectivity for the fluorescence detection of ClO ^- .

3. Anti-interference experiment of DCP-ClO

At the same time, several 5mL EP tubes were taken and similar operations were performed, except that the addition of ^ClO- was replaced by the addition _of various active oxygen and active nitrogen substances. No. 1 to No. 8 correspond to Blank, _H2O2 , ·OH, TBHP, ·OtBu, ^1O2 , ·ON, ^ONOO- , and the concentration of each substance was 1mM. The fluorescence intensity _{( F755nm} ) was measured, and the fluorescence intensity ( _F755nm ) was measured again after adding ^ClO- ( _140μM ). The results are shown in Figure 18(d). It can be seen from Figure 18( _d ) that before the addition of ^ClO- , the fluorescence intensity ( _F755nm ) when only various active oxygen/active nitrogen interfering substances existed in the system was not significantly different from that of the No. 1 blank control group. After the addition of ^ClO- , the fluorescence intensity ( _F755nm ) of all samples increased significantly. This shows that the _presence of the above active oxygen/active nitrogen interfering substances will not affect the fluorescence detection of ^ClO- by DCP- _ClO . Therefore, as a detection reagent, DCP-ClO can effectively avoid the interference of various reactive oxygen/active nitrogen species in its detection of ClO ^- .

4. Performance comparison between DCP-ClO and other ClO ^- fluorescent probes

The fluorescence detection performance of DCP-ClO for ClO ^- was summarized and compared with the performance of the fluorescent probes for detecting ClO ^- in the literature, and the results are shown in Table 1. As can be seen from Table 1, the fluorescence detection response speed of DCP-ClO for ClO ^- reaches or even exceeds the response speed of existing probes. Importantly, when using DCP-ClO for fluorescence detection of ClO ^- , the emission wavelength is in the near-infrared region (755nm) and has a large Stokes shift (100nm), which can effectively reduce the interference of background fluorescence. The detection limit of this method is 3.95×10 ^-8 M, which is better than the sensitivity of the reported near-infrared fluorescence detection method for ClO ^- . Compared with the fluorescent probes described in the literature [3], [11] and [12], the emission wavelength of the probe of the present invention reaches the first near-infrared region (650–900nm), which can effectively avoid background fluorescence interference.

Table 1 Performance comparison of DCP-ClO and ClO ^- fluorescent probes in the literature

References

[1]He M.,Ye M.T.,Wang Z.G.,Liu P.,Li H.Y.,Lu C.F.,Wang Y.Y.,Liang T.,Li H.Y.,Li C.Y.A ratiometric near-infrared fluorescent probe with a largeemission peak shift for sensing and imaging hypochlorous acid.Sensors andActuators B:Chemical,2021,343:130063.

[2]Gao W.J.,Ma Y.Y.,Liu Y.Y.,Ma S.H.,Lin W.Y.Observation of endogenous HClO in living mice with inflammation, tissue injury and bacterialinfection by a near-infrared fluorescent probe.Sensors and Actuators B:Chemical,2021,327:128884 .

[3]Yan FY,Fan KQ,Ma TC,Xu JX,Wang J.,Ma C.Synthesis and spectral analysis of fluorescent probes for Ce ⁴⁺ and OCl ^- ions based onfluorescein Schiff base with amino or hydrazine structure:Application inactual water samples and biological imaging.Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2019,213:254-262.

[4] Shi W.J., Wan Q.H., Yang F., Wang X., Wei Y.F., Lin X.R., Zhang J.Y., Deng R.H., Chen J.Y., Zheng L.Y., Liu F.G., Gao L.Q.A novel TCF-aza-BODIPY-basednear-infrared fluorescent probe for highly selective detection ofhypochlorous acid in living cells.Spectrochimica Acta Part A:Molecular andBiomolecular Spectroscopy,2022,279:121490.

[5]Cho M.,Nguyen V.N.,Yoon J.Y.Simultaneous detection of hypochloriteand singlet oxygen by a thiocoumarin-based ratiometric fluorescent probe.ACSMeasurement Science Au,2022,2(3):219-223.

[6]Zhou Q.,Wang S.Q.,Ran X.Y.,Shen L.Z.,Luo X.L.,Wang G.,Yang H.,WangZ.Y.,Yu X.Q.The water-soluble bicyclic 2-pyridone-based fluorescent probe for fast and selective detection of hypochlorite. Chinese Chemical Letters, https://doi.org/10.1016/j.cclet.2022.107922.

[7] Wang 121481.

[8]Fu J., Hu X., Liang Y., Guo T., Deng F.J., Zhu W.F., Liu M.Y., Wen Y.Q., Zhang of hypochloritein aqueous solution and living cells. Dyes and Pigments, 2022,207:110764.

[9] Xu L.R., Yu J., Wang Y.P., Chen Y.R., Zhang X.L., Han R.B., Jing J., Zhang R.B., Zhang X.L.A mitochondria-targeted fluorescent probe with largeStokes shift for rapid detection of hypochlorite and its application inliving cells. Dyes and Pigments,2022,207:110622.

[10]Chen Y.S.,Zhu Z.Y.,Liu X.Y.,Jiang Y.L.,Shen J.Lysosome-targetingbenzothiazole-based fluorescent probe for imaging viscosity and hypochloritelevels in living cells and zebrafish.Spectrochimica Acta Part A:Molecular andBiomolecular Spectroscopy,2022,275:121141 .

[11] Song X.J., Li C.C., Wang Y., Wang D.H., Liu Z.H. A ratiometric two-photon fluorescence probe for monitoring mitochondrial HOCl produced during the traumatic brain injury process. Sensors and Actuators B: Chemical, 2020, 311: 127895.

[12] Gong Y.J., Lv M.K., Zhang M.L., Kong Z.Z., Mao G.J.A novel two-photonfluorescent probe with long-wavelength emission for monitoring HClO in living cells and tissues. Talanta, 2019,192:128-134.

Example 3: Fluorescence detection of ^ClO- in actual water samples using DCP-ClO as an analytical reagent

In order to verify the practicality of DCP-ClO as an analytical reagent in the fluorescence detection ^of ClO-, the fluorescence detection of ^ClO- in drinking water and tap water was carried out with DCP- ^ClO . At the same time, several 5mL EP tubes were taken, and appropriate volumes of ^ClO- mother solution (10mM) and DCP-ClO mother solution (1mM) were added to drinking water and tap water, respectively, so that the final concentrations of ClO- were 30μM, 60μM and 90μM, respectively, and the final concentration of DCP-ClO was 10μM. After shaking, the system was transferred to a 1cm quartz cell to measure the fluorescence signal of the reaction system. The results of the determination of the content of ^ClO- in the actual water sample are shown in Table 2. It can be seen from Table 2 that the fluorescence detection results of ^ClO- by DCP-ClO are basically consistent with the actual sample spike amount, and the spike recovery rate is within the range allowed by the methodology. The above results prove that DCP-ClO can be used for the fluorescence detection of ^ClO- in actual samples.

Table 2 Detection of ClO ^- in actual samples by DCP-ClO (n=5)

Claims (10)

1. Compound DCP-ClO represented by formula Ⅰ: 2. A method for preparing the compound DCP-ClO of formula I as claimed in claim 1, comprising the following steps: 1) Compound 1 was synthesized using isophorone and malononitrile as raw materials; 2) Using cyclopentanone as a raw material, compound 2 is generated under the action of phosphorus tribromide (PBr ₃ ); 3) reacting compound 2 with 2-hydroxy-4-methoxybenzaldehyde to generate compound 3; 4) Compound 3 is demethylated under the action of boron tribromide (BBr ₃ ) to generate compound 4; 5) Synthesizing DCP-OH using compound 1 and compound 4 as raw materials; 6) reacting the compound DCP-OH with dimethylthiocarbamoyl chloride to obtain the compound DCP-ClO represented by formula I; 3. The preparation method according to claim 2, characterized in that: in step 1), the specific method of synthesizing compound 1 using isophorone and malononitrile as raw materials is: isophorone, malononitrile, piperidine and acetic acid are reacted under nitrogen protection with ethanol as solvent to obtain compound 1; the reaction temperature of the reaction is 90° C. and the reaction time is 6 hours; in the reaction, the molar ratio of isophorone, malononitrile, piperidine and acetic acid is 1:1.1:0.09:0.09; Or, in the step 2), the specific method of using cyclopentanone as a raw material to generate compound 2 under the action of phosphorus tribromide (PBr ₃ ) is: adding phosphorus tribromide (PBr ₃ ) to a mixed solution of DMF and CHCl ₃ at 0°C, stirring for 45 minutes, adding cyclopentanone, and then stirring and reacting at room temperature (25°C) for 16 hours to obtain compound 2; the molar ratio of cyclopentanone to phosphorus tribromide (PBr ₃ ) in the reaction is 1:2.3; the volume ratio of DMF to CHCl ₃ in the mixed solution is 1:4.5; Or, in the step 3), the specific method of reacting compound 2 with 2-hydroxy-4-methoxybenzaldehyde to generate compound 3 is: reacting compound 2, 2-hydroxy-4-methoxybenzaldehyde and cesium carbonate in DMF to obtain compound 3; in the reaction, the molar ratio of compound 2, 2-hydroxy-4-methoxybenzaldehyde and cesium carbonate is 1:1.2:2.5; the reaction is carried out under stirring conditions, the reaction temperature of the reaction is 25°C, and the reaction time is 16h; Or, in the step 4), the specific method of removing the methyl group of the compound 3 under the action of boron tribromide (BBr ₃ ) to generate the compound 4 is as follows: dissolving the compound 3 in anhydrous dichloromethane, slowly adding boron tribromide (BBr ₃ ) in an ice bath, stirring at 0° C. for 1 hour, then slowly warming to room temperature (25° C.), and continuing the reaction for 4 hours to obtain the compound 4; the reaction is carried out under nitrogen protection, and the molar ratio of the compound 3 to boron tribromide (BBr ₃ ) is 1:30; Or, in the step 5), the specific method for synthesizing DCP-OH using compound 1 and compound 4 as raw materials is: dissolving compound 1 and compound 4 in acetonitrile, then adding piperidine and acetic acid in sequence, and reacting under nitrogen protection to obtain compound DCP-OH; in the reaction, the molar ratio of compound 1, compound 4, piperidine and acetic acid is 1:1:5:8.7; the reaction temperature of the reaction is 80°C, and the reaction time is 14h; Or, in the step 6), the specific method of reacting the compound DCP-OH and dimethylthiocarbamoyl chloride to obtain the compound DCP-ClO shown in formula I is: dissolving the compound DCP-OH, dimethylthiocarbamoyl chloride and N,N-diisopropylethylamine (DIPEA) in anhydrous dichloromethane, and reacting under nitrogen protection to obtain the compound DCP-ClO; in the reaction, the molar ratio of the compound DCP-OH, dimethylthiocarbamoyl chloride and DIPEA is 1:12:4.3; the reaction temperature of the reaction is 25°C, and the reaction time is 4h. 4. A fluorescent probe, characterized in that the fluorescent probe is the compound DCP-ClO according to claim 1. 5. A chemical sensor, characterized in that the chemical sensor contains the compound DCP-ClO according to claim 1. 6. Use of the compound DCP-ClO according to claim 1, the fluorescent probe according to claim 4, or the chemical sensor according to claim 5 in detecting ClO ^- ; the use is for non-disease diagnosis purposes. 7. Use of the compound DCP-ClO according to claim 1 in at least one of the following 1)-2): 1) Application as a fluorescent probe; the application is for non-disease diagnosis purposes; 2) Application in the preparation of chemical sensors. 8. The use according to claim 7, characterized in that the fluorescent probe is a fluorescent probe for detecting ClO ^- . 9. The use according to claim 7, characterized in that the chemical sensor is a chemical sensor for detecting ClO ^- . 10. The use according to claim 7, characterized in that the object detected by the fluorescent probe or chemical sensor is water.