CN103923640B - A kind of fluorescence probe and application thereof of benzothiazoles identification hydrogen sulfide - Google Patents

Abstract

A fluorescent probe for recognizing hydrogen sulfide by benzothiazoles and an application thereof belong to the field of fine chemical industry. This fluorescent probe is 2-(2-hydroxyphenyl)benzothiazole derivative; mix 2-(2-hydroxyphenyl)benzothiazole with potassium carbonate and 2,4-dinitrobromobenzene in proportion , and finally purified by silica gel chromatography to obtain the fluorescent probe. The fluorescent probe and the corresponding hydrogen sulfide content detection process will not be interfered by the matrix and impurities of the biological system, and can be used for the quantitative determination of the hydrogen sulfide content in various biological systems. This should probe has high specificity and can be hydrolyzed after specific cyclization with hydrogen sulfide, that is, the hydrolyzed product of ether bond breaking; it is cheap and easy to obtain, and can be obtained by chemical synthesis, and the synthesis process is simple and easy; it has high sensitivity and good Fluorescence emission spectrum characteristics (450-500nm), quantitative determination of hydrogen sulfide by drawing a standard curve.

Description

A kind of fluorescent probe and its application of benzothiazole recognition hydrogen sulfide

technical field

The invention relates to a fluorescent probe for recognizing hydrogen sulfide by benzothiazoles and an application thereof, belonging to the field of fine chemical industry.

Background technique

For centuries, hydrogen sulfide (H ₂ S) was considered to be a toxic substance produced by geological activities or microbial action. This colorless, flammable gas has a disgusting smell of rotten eggs and is a strong irritant to the eyes and respiratory system of mammals. Excessive inhalation of hydrogen sulfide gas can lead to loss of consciousness, respiratory failure, cardiac arrest and other physiological reactions, and excessive absorption can lead to death. On the other hand, more and more studies have begun to challenge this traditional concept: mammals can produce hydrogen sulfide under controlled conditions, and it is believed that oxygen sulfide plays an important role in maintaining normal physiological functions. Hydrogen sulfide can be produced by non-enzymatic actions in aspirin mammals, including the release of sulfur pools in the body and the metabolism of polysulfide compounds. It can also be catalyzed by a variety of enzymes, including cystathionine-polymerase (CBS), cystathionine-lyase (CSE), cysteine transferase and mercaptopyruvate transsulfurase. These enzymes can catalyze sulfur-containing biomolecules such as cysteine and homocysteine to produce hydrogen sulfide through different reactions. These enzymes appeared in the tissues of various organs such as the heart, vasculature, brain, kidney, lung, and pancreas, indicating that oxygen sulfide is widely distributed in mammals.

In mammals, hydrogen sulfide can undergo thiol reaction with downstream proteins through post-translational cysteine and connect to the iron center of heme. This process will control multiple physiological responses, including ischemia-reperfusion-induced injury, vasodilation, cell death, angiogenesis, neuromodulation, inflammation therapy, insulin signaling, and oxygen stress, among others. In addition, hydrogen sulfide also acts as an antioxidant or scavenger for reactive oxygen species. However, the abnormal concentration level of H ₂ S has been confirmed to be related to many diseases. These pioneering experiments have shown that hydrogen sulfide is an important physiological regulating gas and not just a pollutant. The complex physiological roles and potential therapeutic applications of hydrogen sulfide have prompted researchers to propose new methods for monitoring the production, circulation and consumption of hydrogen sulfide in different cells, tissues and organs.

Traditional methods for the detection of hydrogen sulfide, including colorimetry, polarographic sensing and gas chromatography, usually cause damage to the sample to be tested, or cannot detect sulfide in organisms at all. Fluorescent molecular probes provide an attractive method for the detection of hydrogen sulfide and other small biomolecules in biological systems due to their membrane permeability and high selectivity. Fluorescence analysis will provide important biological information for studying the physiological function of hydrogen sulfide. In the past two years, the reports on the optical detection of hydrogen sulfide have attracted great attention of researchers.

The invention provides a class of 2-(2-hydroxyphenyl)benzothiazole derivatives as probes for specifically recognizing hydrogen sulfide (H ₂ S), which can generate hydrolyzed products with fluorescent properties after hydrolysis with hydrogen sulfide . The reaction has the characteristics of high selectivity, high sensitivity and rapid response.

Contents of the invention

The purpose of the present invention is to provide a specific fluorescent probe for recognizing hydrogen sulfide. The probe itself has weak fluorescence, and the product reacted with hydrogen sulfide (H ₂ S) has extremely strong fluorescence properties. The probe reaction can be used to quantitatively evaluate the content of hydrogen sulfide (H ₂ S) in various biological samples.

The technical solution of the present invention is: a benzothiazole-based fluorescent probe for recognizing hydrogen sulfide, the fluorescent probe is a 2-(2-hydroxyphenyl) benzothiazole derivative, and its structural formula is as follows:

The preparation method of described fluorescent probe comprises the steps:

(1) According to the molar ratio of 2-(2-hydroxyphenyl)benzothiazole:2,4-dinitrobromobenzene:potassium carbonate is 1:1~2:2~3, add it into the reaction flask, add Acetonitrile, control the reaction temperature at 40~80°C, stir for 8-12h;

(2) The solvent was removed from the above reaction solution under reduced pressure, and the residual solid was purified by silica gel chromatography, and eluted with an eluent with a volume ratio of ethyl acetate:n-hexane of 1:3 to obtain a fluorescent probe

The fluorescent probe is applied to quantitative evaluation of hydrogen sulfide content in biological samples. As a specific probe for hydrogen sulfide, the probe undergoes a hydrolysis reaction, and the content of hydrogen sulfide in the cell is determined by quantitatively detecting the fluorescence intensity of the hydrolyzed product; the specific determination method is:

In the system, 2-(2-hydroxyphenyl)benzothiazole derivatives are used as specific probes; the probe concentration is 1/~10 μM; common buffers such as PBS or Tris-HCl: dimethyl sulfoxide mixed solution (Volume ratio 7:3), the reaction temperature is between 20°C and 60°C, preferably 37°C is the optimal reaction time; the pH of the reaction system is between 5.5 and 10.5, preferably pH 7.4 is the optimal reaction pH value ; The reaction time is 5-120 minutes; The fluorescence intensity of the hydrolyzate is measured as the evaluation index of the hydrogen sulfide content.

The probe itself has weak fluorescence, and its hydrolyzed products have extremely strong fluorescence. Fluorescence detectors can be used to realize rapid and sensitive detection of products and substrates; the fluorescence detection conditions are: excitation wavelength 300nm, fluorescence emission spectrum at 450~500nm detection.

The beneficial effects of the present invention are: the fluorescent probe is 2-(2-hydroxyphenyl)benzothiazole derivative; 2-(2-hydroxyphenyl)benzothiazole sodium salt and potassium carbonate, 2 , mixed with 4-dinitrobromobenzene, added N, N-dimethylformamide, heated, and finally purified by silica gel chromatography to obtain a fluorescent probe. The fluorescent probe and the corresponding hydrogen sulfide content detection process will not be interfered by the matrix and impurities of the biological system, and can be used for the quantitative determination of the hydrogen sulfide content in various biological systems. This should probe has high specificity and can be hydrolyzed after specific cyclization with hydrogen sulfide, that is, the hydrolyzed product of ether bond breaking; it is cheap and easy to obtain, and can be obtained by chemical synthesis, and the synthesis process is simple and easy; it has high sensitivity and good Fluorescence emission spectrum characteristics (450-500nm), quantitative determination of hydrogen sulfide by drawing a standard curve.

Description of drawings

Fig. 1 is a ¹ H-NMR spectrum of 2-(2(2,4-dinitrophenoxy))benzothiazole.

Fig. 2 is a ¹³ C-NMR spectrum of 2-(2(2,4-dinitrophenoxy))benzothiazole.

Figure 3 is the high resolution mass spectrum of 2-(2(2,4-dinitrophenoxy))benzothiazole.

Fig. 4 is the result of fluorescence change after the fluorescent probe reacts with different substances.

Fig. 5 is the fluorescence intensity change curve after the fluorescent probe reacts with different concentrations of hydrogen sulfide.

Fig. 6 is the relationship between the fluorescence intensity change and the time when the fluorescent probe reacts with hydrogen sulfide.

Figure 7 is a confocal imaging diagram.

detailed description

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

Chemical synthesis of embodiment 12-(2(2,4-dinitrophenoxy))benzothiazole

(1) Dissolve 0.5mmol of 2-(2-hydroxyphenyl)benzothiazole, 0.625mmol of potassium carbonate and 0.5mmol of 2,4-dinitrobromobenzene in 10mL of acetonitrile, and react at 80°C for 4h;

(2) The reaction solution is subjected to vacuum distillation;

(3) The solid was purified by silica gel chromatography, and eluted with ethyl acetate-n-hexane (1:3 v/v) to obtain a light yellow solid. The characterization results are shown in Figure 1, Figure 2 and Figure 3. ¹ HNMR(400MHz,DMSO)δ8.98(d,J=2.8Hz,1H),8.50(dd,J=7.9,1.6Hz,1H),8.42(dd,J=9.3,2.8Hz,1H),8.11 (d,J=8.0Hz,1H),8.04(d,J=8.1Hz,1H),7.79–7.68(m,1H),7.66–7.51(m,2H),7.47(dd,J=14.4,7.5 Hz,2H),7.24(d,J=9.3Hz,1H).13CNMR(100MHz,CDCl3)δ160.87,155.66,152.67,150.68,141.78,139.54,135.50,132.51,131.30,128.99,127.45,126.60,121 , 123.46, 122.25, 122.13, 121.52, 117.80. HRMS calcd for C16H14NO5+([M+H]+) 394.0492, found 394.0501.

Example 22-(2(2,4-dinitrophenoxy))benzothiazole selectivity for different substances

(1) Prepare 99 μl metabolic reaction system in advance, including PBS buffer (10 mM) at pH 7.4: dimethyl sulfoxide (volume ratio 7:3), fluoride ion (100 μM), chloride ion (50 μM), bromide ion ( 100μM), iodide ion (100μM), sodium ion (100μM), potassium ion (100μM), calcium ion (100μM), magnesium ion (100μM), nitrate ion (100μM), sodium hydrosulfide (100μM);

(2) Add 1 μl of 2-(2(2,4-dinitrophenoxy))benzothiazole at a final concentration of 10 μM to the reaction system to initiate the reaction;

(3) After 30 minutes, perform fluorescence detection (λ _Ex =300nm, λ _Em =458nm); calculate the fluorescence intensity in each system (see Figure 4).

Example 32-(2(2,4-dinitrophenoxy))benzothiazole and hydrogen sulfide concentration linear relationship

(1) Prepare 99μl metabolic reaction system in advance, including PBS buffer (10mM) at pH 7.4: dimethyl sulfoxide (volume ratio 7:3), hydrogen sulfide (0-100μM), react at 37°C for 30 minute;

(2) Add 1 μl of 2-(2(2,4-dinitrophenoxy))benzothiazole at a final concentration of 10 μM to the reaction system to initiate the reaction;

(3) After 30 minutes, perform fluorescence detection (λ _Ex =300nm, λ _Em =458nm); calculate the fluorescence intensity in each system, and establish a standard curve of fluorescence intensity and hydrogen sulfide concentration (see Figure 5); the standard curve is y=3695.9x -373.09, R2=0.9931, where, y represents the fluorescence intensity at 458nm, and x represents the concentration of sodium hydrosulfide (the linear relationship between the concentration of hydrazine is satisfied between 0uM and 100uM).

Example 42-(2(2,4-Dinitrophenoxy))benzothiazole as a probe reacts with hydrogen sulfide as a relationship between fluorescence intensity change and time

(1) Prepare 99 μl metabolic reaction system in advance, including PBS buffer (10 mM) at pH 7.4: dimethyl sulfoxide (volume ratio 7:3), hydrogen sulfide (200 μM), and react at 37°C for 30 minutes;

(2) Add 1 μl of 2-(2(2,4-dinitrophenoxy))benzothiazole at a final concentration of 10 μM to the reaction system to initiate the reaction;

(3) After 30 minutes, perform fluorescence detection (λ _Ex =300nm, λ _Em =458nm); calculate the fluorescence intensity in each system, and establish a linear relationship curve between fluorescence intensity and hydrogen sulfide reaction time (see Figure 6).

Example 5 Quantitative determination of hydrogen sulfide content in human lung adenocarcinoma A549 cells

(1) The human lung adenocarcinoma A549 cell line was cultured on coverslips, the medium used was DMEM medium (containing 10% calf serum) and 100 μg/ml double antibody, and the culture environment was 5% carbon dioxide culture at 37°C in the box;

(2) Before use, the adherent cells were washed with serum-free DMEM medium for 3 times, and 2-(2(2,4-dinitrophenoxy))benzothiazole was added at a final concentration of 10 μM, at 37 Incubate at ℃ for 40 minutes;

(3) After that, wash with PBS buffer 3 times. Observe the cells under a laser confocal microscope, and display the content in the cells through the fluorescence distribution position and intensity (see Figure 7).

Embodiment 6 Quantitative determination of hydrogen sulfide content in human blood

(1) Add 20 μl to 380 μl of 10% normal human plasma diluted with PBS: dimethyl sulfoxide solution (volume ratio 7:3) and add 20 μl of final hydrogen sulfide concentrations of 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300μM hydrogen sulfide solution;

(2) Add 1 μl of 2-(2(2,4-dinitrophenoxy))benzothiazole at a final concentration of 10 μM to the reaction system to initiate the reaction;

(3) Perform fluorescence detection (λ _Ex =300nm, λ _Em =458nm); calculate the signal-to-noise ratio S/N>10, and detect the fluorescence intensity signals in plasma with different hydrogen sulfide contents.

Claims (2)

1. A fluorescent probe for benzothiazole recognition of hydrogen sulfide, characterized in that: the fluorescent probe is 2-(2-hydroxyphenyl) benzothiazole sodium salt derivative, and its structural formula is as follows: The preparation method of described fluorescent probe comprises the steps: (1) According to the molar ratio of 2-(2-hydroxyphenyl)benzothiazole:2,4-dinitrobromobenzene:potassium carbonate is 1:1~2:2~3, add it into the reaction flask, add Acetonitrile, control the reaction temperature at 40~80°C, stir for 8-12h; (2) The solvent was removed from the above reaction solution under reduced pressure, and the residual solid was purified by silica gel chromatography, and eluted with an eluent with a volume ratio of ethyl acetate:n-hexane of 1:3 to obtain a fluorescent probe. 2. a kind of fluorescent probe of benzothiazole class recognition hydrogen sulfide according to claim 1, it is characterized in that: described fluorescent probe is applied to the quantitative evaluation of hydrogen sulfide content in the biological sample, and hydrogen sulfide concentration is 0- 100μM, establish a standard curve of fluorescence intensity and hydrogen sulfide concentration, the standard curve is y=3695.9x-373.09, R2=0.9931, where y represents the fluorescence intensity at 458nm, and x represents the concentration of sodium hydrogen sulfide.