CN116903601A - Poly hydrogen sulfide near infrared chemiluminescence probe, preparation method and application thereof - Google Patents

Abstract

The invention relates to a polyhydrogen sulfide near-infrared chemiluminescent probe, a preparation method and its application. The structural formula of the chemiluminescent probe CL-SN is shown in formula (I). The near-infrared chemiluminescence probe CL‑SN has the advantages of high sensitivity, low detection limit, high signal-to-noise ratio, high selectivity, and non-invasive real-time imaging; the probe CL‑SN realizes the chemiluminescence of polyhydrogen sulfide in H9c2 cells. Imaging can be further applied to the in vivo level of mice, enabling the detection of polyhydrogen sulfide at the in vivo level. The probe can be used to detect polyhydrogen sulfide in complex environments within organisms, providing an effective detection tool for changes in polyhydrogen sulfide levels in diseases.

Description

Poly hydrogen sulfide near infrared chemiluminescence probe, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a hydrogen polysulfide near-infrared chemiluminescent probe, a preparation method and application thereof.

Background

Hydrogen sulfide (H) ₂ S) is a third gas signaling molecule following Nitric Oxide (NO) and carbon monoxide (CO). H ₂ S changes the structure and function of target protein mainly through sulfhydrylation modification of cysteine residue, and influences the activity of key receptor and signal path so as to play a physiological role. Polyhydrosulfide (H) ₂ S _n N.gtoreq.2) is H ₂ Oxidation products of S. At H ₂ In the S-related physiological process, the hydrogen polysulfide plays an important regulatory role. The hydrogen polysulfide can promote signal transduction between cells, is an important redox signal, and has the effects of cytoprotection, anti-inflammation, anticancer, antioxidation and the like.

The detection method of the hydrogen polysulfide mainly comprises the following steps: ultraviolet/visible light absorption spectrometry, mass spectrometry, chromatography, fluorescent probe methods. However, the first three methods require pretreatment of cells and tissues. The method has the defects of long detection time, high cost, complex operation and the like, and cannot detect the hydrogen polysulfide in real time and in situ. In order to explore the mechanism of action of hydrogen polysulfide in organisms, accurately detect the hydrogen polysulfide level, it is required to establish an effective real-time, dynamic and visual accurate detection technique.

The fluorescent probe has the advantages of noninvasive real-time monitoring, high sensitivity and selectivity, good biocompatibility and the like. Thus, many studies for detecting hydrogen polysulfide based on a fluorescent probe method have been reported. Although some prior hydrogen polysulfide fluorescent probes have advanced, noninvasive in-vivo imaging using these fluorescent probes is still affected by the light penetration depth of fluorescence of biological tissues under external light excitation, photobleaching and high tissue autofluorescence interference.

Unlike conventional fluorescence imaging, chemiluminescence (chemiluminesce) is a phenomenon in which light is generated by chemical excitation in a chemical reaction. Because no real-time excitation of an external light source is needed, chemiluminescence has little autofluorescence interference, and can realize higher signal-to-noise ratio and improve the sensitivity of in-vivo imaging. 1, 2-dioxetane (1, 2-dioxetanone) is the most common chemiluminescent substrate, and the hydroxyl groups in its structure facilitate structural modification, thereby developing a variety of chemiluminescent probes. A series of chemiluminescent probes have been successfully used to detect biologically active substances such as beta-galactosidase, hydrogen peroxide, and cysteine.

Near-infrared (NIR) imaging technology is a major trend in research in the field of detection of small bioactive molecules. However, the emission wavelength of the 1, 2-dioxetane is generally below 600nm, and the generated photons are easily absorbed by tissues, so that the detected optical signals are weakened. Dicyanopyran substituted 1, 2-dioxetane derivatives developed by Shbat et al have an emission that can red shift to 700nm. Therefore, the near infrared chemiluminescent probe is used for detecting the hydrogen polysulfide, which is beneficial to imaging deep tissues and further improves the detection sensitivity of the deep tissues.

Disclosure of Invention

The invention aims to provide a near infrared chemiluminescence probe for hydrogen polysulfide, and a preparation method and application thereof based on the prior art.

The aim of the invention can be achieved by the following measures:

a near infrared chemiluminescence probe of hydrogen polysulfide has a chemical structure shown in a formula (I),

a preparation method of a near infrared luminescent probe of hydrogen polysulfide comprises the following synthetic routes:

in the present invention, the reaction is carried out using 1g of the compound as a starting material, wherein the preparation method of 1g of the compound can be referred to as CN115417863A.

In the preparation method, the specific reaction steps comprise: the compound 1g and 2- (benzoylthio) benzoic acid are subjected to condensation reaction, and then the reaction product is catalyzed by methylene blue under the condition of introducing oxygen to obtain the compound of the formula (I).

In a preferred embodiment, 1g of the compound is dissolved in a solvent together with 2- (benzoylthio) benzoic acid, EDC and DMAP are then added, and the mixture is stirred at 20-30 ℃ for condensation reaction for 0.2-3h; then under the condition of introducing oxygen and under the irradiation of yellow light, the reaction is catalyzed by methylene blue, and the compound shown in the formula (I) is obtained. The reaction temperature in the condensation reaction is room temperature, the reaction time is preferably 1h, and the reaction solvent is preferably dichloromethane. In the catalytic reaction, the reaction temperature is 20-30 ℃, preferably 25 ℃, and the reaction time is 0.2-3h, preferably 1h.

In a preferred embodiment, the molar ratio of compound 1g to 2- (benzoylthio) benzoic acid is 1:1 to 2, preferably 1:1.2 to 1.8, more preferably 1:1.5.

in another preferred embodiment, the molar ratio of DMAP to EDC is 1:2 to 4, preferably 1:2.8 to 3.2, more preferably 1:3.0.

in another preferred embodiment, the molar ratio of compound 1g to EDC is 1:0.5 to 2.5, preferably 1:1 to 2, more preferably 1:1.5.

in a preferred process for the preparation of a hydrogen polysulfide near infrared chemiluminescent probe, 1g is condensed with 2- (benzoylthio) benzoic acid, then EDC and DMAP are added in the presence of oxygen to react, and then the compound of formula (I) is obtained by methylene blue catalysis.

The near infrared chemiluminescence probe of the invention can be applied to chemiluminescence imaging for detecting the hydrogen polysulfide in cells or mice.

The near infrared chemiluminescence probe of the invention can be applied to the detection of endogenous hydrogen polysulfide in cells or mice.

The near infrared chemiluminescence probe of the invention can be applied to the aspect of chemiluminescence imaging detection of endogenous hydrogen polysulfide in cells or mice.

The near infrared chemiluminescence probe for the hydrogen polysulfide can be applied to quantitative detection of the hydrogen polysulfide.

The near infrared chemiluminescence probe of the invention can be applied to the aspect of detecting the physiological and pathological mechanism or signal transduction path of the hydrogen polysulfide in an organism.

The invention relates to a near infrared chemiluminescence probe of hydrogen polysulfide, which has a chemical structure shown in a formula (I), and is also called a probe CL-SN.

The invention has the beneficial effects that:

the invention designs and synthesizes a near infrared chemiluminescent probe CL-SN for identifying hydrogen polysulfide by taking dicyanopyran substituted adamantane-dioxetane as a near infrared chemiluminescent substrate and 2- (benzoyl mercapto) benzoate as an identification group. Probes CL-SN and Na ₂ S ₄ The reaction products of (2) are characterized by high resolution mass spectrometry to determine the probe reaction mechanism. The probe has good selectivity and high sensitivity (detection limit 39 nM). The probe CL-SN also realizes the chemiluminescent imaging detection of H9c2 cell-level hydrogen polysulfide. More importantly, the probe CL-SN realizes chemiluminescent imaging detection of the living body level hydrogen polysulfide. The method can provide a visual detection method for the physiological and pathological mechanism of the hydrogen sulfide in the organism and the signal transduction path, and has important significance for revealing the physiological and pathological mechanism of the hydrogen sulfide in the human body.

Drawings

FIG. 1 shows probes CL-SN and Na ₂ S ₄ Chemiluminescent kinetics measurements of the reaction;

in the figure, the upper line is CL-SN, and the lower line is CL-SN+Na ₂ S ₄ 。

FIG. 2 shows the probe CL-SN and different concentrations of Na ₂ S ₄ A chemiluminescent response result map of (2);

the probe CL-SN and Na at different concentrations are shown ₂ S ₄ (0-500. Mu.M) in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37 ℃Chemiluminescent response after 1h incubation. Insert diagram: probe CL-SN chemiluminescence intensity and Na ₂ S ₄ Linear relationship of concentrations (0, 1,2, 4, 6, 8, 12, 14, 16, 18, 20 μm).

FIG. 3 is the selectivity of probe CL-SN to hydrogen polysulfide;

in the figure, probe CL-SN (10 μm) was incubated in PBS buffer (20 mm, ph= 7.4,10% DMSO,10% FBS) for 1h at 37 ℃, chemiluminescent intensity for each thiol, 1.blank; gsh (1 mM); gsh (10 mM); gssg (1 mM); 5.CH ₃ SSSCH ₃ (1mM)；6.Hcy(100μM)；7.S ₂ O ₃ ^2- (1mM)；8.SO ₄ ^2- (1mM)；9.S ₂ O ₄ ^2- (1mM)；10.HSO ₃ ^- (1mM)；11.Cys(1mM)；12.L-Cys(1mM)；13.D-Cys(1mM)；14.Na ₂ S(100μM)；15.S ₈ (1mM)；16.Na ₂ S ₄ (1mM))。

FIG. 4 is a sample of probe CL-SN vs. Na ₂ S ₄ Selectivity of (2);

it was incubated in PBS buffer (20 mm, ph= 7.4,10% DMSO,10% FBS) at 37 ℃ for 1h, the chemiluminescent intensity of probe CL-SN (10 μm) on active oxygen and active nitrogen, 1.blank;2.H ₂ O ₂ (100μM)；3.ClO ^- (100μM)；4. ^t BuOOH(100μM)；5.·OH(100μM)；6. ¹ O ₂ (100μM)；7.O ^2- (100μM)；8.NO ₃ ^2- (100μM)；9.NO ^2- (100μM)；10.ONOO ^- (100μM)；11.NO(100μM)；12.Na ₂ S ₄ (500μM))。

FIG. 5 shows the probe CL-SN vs. Na ₂ S ₄ Selectivity of (2);

it was incubated in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37℃for 1h, and the chemiluminescent intensity of probe CL-SN (10. Mu.M) on inorganic salt ions was 1.blank; zn (Zn) ²⁺ (1mM)；3.Na ⁺ (1mM)；4.K ⁺ (1mM)；5.Mg ²⁺ (1mM)；6.Cu ²⁺ (1mM)；7.Ca ²⁺ (1mM)；8.Fe ³⁺ (1mM)；9.Fe ²⁺ (1mM)；10.Al ³⁺ (1mM)；11.Cl ^- (1mM)；12.I ^- (1mM)；13.F ^- (1mM)；14.HPO ₄ ^2- (1mM)；15.H ₂ PO ₄ ^- (1mM)；16.CO ₃ ^2- (1mM)；17.HCO ₃ ^- (1mM)；18.Na ₂ S ₄ (500μM))。

FIG. 6 is a sample of probe CL-SN vs. Na ₂ S ₄ Selectivity of (2);

it was incubated in PBS buffer (20 mM, pH= 7.4,10% DMSO,10% FBS) at 37℃for 1h, probe CL-SN (10. Mu.M) was 1.blank for chemiluminescent intensity of amino acids; glu (1 mM); trp (1 mM); tyr (1 mM); ser (1 mM); arg (1 mM); phe (1 mM); his (1 mM); ala (1 mM); val (1 mM); pro (1 mM); gly (1 mM); na. Na ₂ S ₄ (500μM))。

FIG. 7 pH vs. probes CL-SN and Na ₂ S ₄ Influence of the reaction;

it was incubated at 37℃for 7h, probe CL-SN (10. Mu.M) and Na ₂ S ₄ (500. Mu.M)) in different pH buffers (20 mM, pH 4.0, 4.5, 5.0, 6.0, 6.5, 7.0, 7.4, 7.5, 8.0, 8.5 and 9.0,10% DMSO,10% FBS). The upper line in the figure is CL-SN+Na ₂ S ₄ The lower line is CL-SN.

FIG. 8 is a stability test result of the probe CL-SN;

it was incubated at 37℃for 1h and probe CL-SN (10. Mu.M) was chemiluminescent at 37℃in PBS buffer (20 mM, pH=7.4, 10% DMSO,10% FBS). The upper dot in the figure shows CL-SN in PBS buffer, and the lower dot shows PBS buffer.

FIG. 9 is a graph showing the effect of probe CL-SN on H9c2 cell viability;

in the diagram a: survival of H9c2 cells incubated with CL-SN (0, 5,10,20, 50. Mu.M) for 24H cells; in the diagram B: viability of H9c2 cells incubated with CL-SN (10. Mu.M) for various times (0, 6,12,18, 24H).

FIG. 10 is a cytoluminescent image of H9c2 cell exogenous hydrogen polysulfide;

in the figure, a is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37 ℃. In the figure, B is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37℃and then with Na ₂ S ₄ (25. Mu.M) incubating for 10min. In the figure, C is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37℃and then with Na ₂ S ₄ (50. Mu.M) for 10min. In the figure, D is: cells were pretreated with NMM (1 mmol) for 1h, then incubated with CL-SN (10. Mu.M) for 30min, and then with Na ₂ S ₄ (50. Mu.M) for 10min. In the figure, E is: the total photon flux of chemiluminescence was quantified for each set of cell areas. Data are expressed as mean ± SD (n=3). ^*** Comparison with group a P<0.001。 ^### Comparison with group C P<0.001。

FIG. 11 is a cytochemiluminescent imaging of H9c2 cells endogenous to the hydrogen polysulfide;

in the figure, a is: cells were incubated with CL-SN (10. Mu.M) for 30min at 37 ℃; in the figure, B is: cells were incubated with LPS (1. Mu.g/mL) at 37℃for 16h, then with CL-SN (10. Mu.M) for 30min; in panel C, cells were incubated with DL-propylglycine (PAG, 200. Mu.M) at 37℃for 30min, followed by addition of LPS (1. Mu.g/mL) for 16h and then CL-SN (10. Mu.M) for 30min; in the figure, D is: quantitatively measuring the total photon flux of chemiluminescence of each group of cell areas; ^*** comparison with group a P<0.001。 ^### Comparison with group C P<0.001。

FIG. 12 is a sample of the detection of in vivo horizontal hydrogen polysulfide by probe CL-SN;

in the figure, a is: mice were injected intraperitoneally with probe CL-SN (100 μΜ,100 μl, DMSO: physiological saline = 1:9); in the figure, B is: abdominal injection of Na into mice ₂ S ₄ (2 mM, 100. Mu.L of physiological saline) for 10min, and then intraperitoneally injecting probe CL-SN (100. Mu.M, 100. Mu.L, DMSO: physiological saline=1:9); in the figure, C is: abdominal injection of Na into mice ₂ S ₄ (4 mm,100 μl of physiological saline) for 10min, and then intraperitoneally injecting probe CL-SN (100 μΜ,100 μl, DMSO: physiological saline=1:9); in the figure, D is: mice were intraperitoneally injected with LPS (10 μg/mL,100 μl of saline) for 24h, followed by CL-SN (100 μΜ,100 μl, DMSO: saline=1:9); in the figure, E is: mice were pre-treated with DL-propylglycine (2 mm,100 μl of physiological saline) for 30min and then were intraperitoneally injected with LPS (10 μg/mL,100 μl of physiological saline) for 24h, and CL-SN (100 μl, DMSO: physiological saline=1:9) was intraperitoneally injected; f in the figure is: the total photon flux of the abdominal chemiluminescence of each group of mice was quantitatively determined. Data as mean ± SD (n=3); ^*** comparison with group a P<0.001。 ^### Comparison with group D P<0.001。

FIG. 13 is a probe CL-SN+Na ₂ S ₄ HRMS of the reaction product; wherein, (M-H) ^- :403.0486,found,403.0486。

Fig. 14 is probe CL-SN: ¹ H NMR(400MHz,CDCl ₃ )。

fig. 15 is probe CL-SN: ¹³ C NMR(100MHz,CDCl ₃ )。

FIG. 16 is HRMS of probe CL-SN; wherein (M+Na) ^- C ₄₆ H ₃₅ ClN ₂ O ₇ S,817.1746；found817.1702。

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

1.1 preparation of solution:

(1) Preparing a probe CL-SN (namely a compound of a formula (I)) solution; under the dark environment, the probe CL-SN is dissolved in DMSO to prepare a 1mM probe solution.

(2)Na ₂ S ₄ Preparation of solutions (as a source of hydrogen polysulfide): na (Na) ₂ S ₄ A solution of 20mM sodium polysulfide was prepared in PBS buffer (20 mM, pH=7.4, 10% DMSO).

(3) Solutions of other biological analytes, including active sulfur species (RSS, including HSO ₃ ^- ；SO ₃ ^2- ；S ₂ O ₃ ^2- ；SO ₄ ^2- ；S ₂ O ₄ ^2- ；GSSG；S ₈ ；CH ₃ SSSCH ₃ The method comprises the steps of carrying out a first treatment on the surface of the Cys; hcy; GSH), reactive oxygen species (ROS, including H ₂ O ₂ ；ClO ^- ；O ₂ ^- ；·OH； ^t BuOOH； ¹ O ₂ ) Reactive nitrogen (RNS, including NO; NO (NO) ₂ ^- ；ONOO ^- ；NO ₃ ^- ) Inorganic salt ion (K) ⁺ ；Ca ²⁺ ；Na ⁺ ；Mg ²⁺ ；Fe ³⁺ ；Zn ²⁺ ；Cu ²⁺ ；CO ₃ ^2- ；HCO ₃ ^- ；Cl ^- ；I ^- ；HPO ₄ ^2- ；H ₂ PO ₄ ^- ) Amino acids (Glu; trp; tyr; ser; arg; phe; his; ala; met; leu; val; pro; gly), and the like.

1.2 cells:

species and strain: rat cardiomyocyte H9c2 cell line. The source is as follows: cell bank of China academy of sciences.

1.3 animals:

species and strain; healthy male Kunming mice, weighing 20-25g. The source is as follows: xuzhou university of medical science laboratory animal center.

EXAMPLE 1 Synthesis of Compound of formula (I) (i.e., compound 1 CL-SN)

Compound 1g (52.2 mg,0.1 mmol), 2- (benzoylthio) benzoic acid (39 mg,0.15 mmol) was dissolved in 10mL of dichloromethane, EDC (29 mg,0.15 mmol) and DMAP (6.1 mg,0.05 mmol) were then added and the reaction stirred at room temperature for 1h. After completion of the reaction, the reaction mixture was washed with distilled water (100 mL) and extracted with methylene chloride (3X 100 mL). The organic layer was treated with anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure to remove the solvent. The crude product was purified by column chromatography on silica (silica, PE: EA,20:1 v/v) to give 50mg of a yellow solid. The yellow solid was dissolved in 20mL of methylene chloride, methylene blue (3 mg,0.1 mmol) was added thereto, oxygen was introduced, and the reaction was performed for 1 hour under reduced pressure to remove the solvent. The crude product was purified by column chromatography on silica gel (silica, PE: EA,17:3 v/v) to give 90mg of a white solid. The yield thereof was found to be 30%.

TLC(silica,PE:EA,17:3v/v)：Rf＝0.45。 ¹ H NMR(400MHz,CDCl ₃ ):δ8.87-8.80(m,

1H),8.50-8.31(m,2H),8.16-8.11(m,1H),7.88-7.58(m,8H),7.49-7.37(m,2H),7.24-7.20(m,2H),6.98-6.80(m,2H),3.27-3.24(m,3H),3.03(s,1H),2.27-2.24(m,1H),2.07-1.63(m,12H). ¹³ C NMR(100MHz,CDCl ₃ ):δ164.01,161.85,156.15,153.36,152.99,152.55,152.14,135.26,134.93,134.71,134.63,131.26,130.95,130.72,130.60,129.10,128.16,127.44,126.24,126.14,125.87,124.93,123.18,118.75,118.66,117.72,117.62,116.69,116.43,115.57,115.29,111.70,108.32,105.57,96.56,62.36,49.94,36.65,33.99,33.73,32.31,31.65,29.78,26.24,25.87,20.58.HRMS(ESI ⁺ ):(M+Na) ^- C ₄₆ H ₃₅ ClN ₂ O ₇ S,817.1746；found 817.1702.

EXAMPLE 2 detection Performance study of chemiluminescent Probe CL-SN on Hydrogen polysulfide

Under a dark environment, the probe CL-SN is dissolved in DMSO to prepare a mother solution concentration (1 mM). Na (Na) ₂ S ₄ (as a source of hydrogen polysulfide) in PBS buffer (20 mM, pH= 7.4,10% DMSO) to make up a 20mM sodium polysulfide solution. Probe CL-SN (final concentration 10. Mu.M) and Na ₂ S ₄ (final concentration 500. Mu.M) chemiluminescent signal was detected in PBS (10 mM,10% FBS, pH 7.4) at 37 ℃. Each set of data was run at least three times in parallel and the results were expressed as mean±sd.

Test conditions: chemiluminescent in vitro experiments were performed using a Varioskan LUX multi-function microplate reader for chemiluminescent kinetics measurements. Observation Probe CL-SN (10. Mu.M) and CL-SN (10. Mu.M) +Na ₂ S ₄ (500. Mu.M) chemiluminescent response was measured over several hours. As can be seen from fig. 1, as the reaction time increases, the chemiluminescent intensity increases and decreases, and at 15min, the chemiluminescent intensity peaks; around 2h, there is still a strong chemiluminescent signal.

Example 3 chemiluminescent probe CL-SN detection limit determination of Hydrogen polysulfide

The calculation method comprises the following steps: the standard deviation of the blank, i.e., the chemiluminescent intensity of the detection probe CL-SN in the HEPES buffer system, was measured and calculated after 10 repetitions. Then the probe CL-SN and Na are measured ₂ S ₄ Linear equation of reaction in the range of 0-20. Mu.M. The calculation formula of the detection limit is as follows: 3 sigma/k. k represents probe and Na ₂ S ₄ Slope of the linear equation of the reaction, σ, represents standard deviation of the blank.

As a result, as shown in FIG. 2, probe CL-SN was reacted with Na at various concentrations ₂ S ₄ After incubation (0-500. Mu.M), the chemiluminescent intensity follows Na ₂ S ₄ The increase in concentration was gradually increased (28-fold). And the chemiluminescent intensity shows good linear relation in the range of 0-20 mu M, and the detection limit of the probe CL-SN is 39nM. Experimental results show that the probe CL-SN has good detection sensitivity, and can quantitatively detect the hydrogen polysulfide.

Solutions of other biological analytes were also tested for selectivity of the probe for hydrogen polysulfide.

As shown in FIGS. 3 to 6, the probes CL-SN and Na ₂ S ₄ Incubation produces a distinct chemiluminescent signal, but with other active sulfur species (RSS, including HSO ₃ ^- ；SO ₃ ^2- ；S ₂ O ₃ ^2- ；SO ₄ ^2- ；S ₂ O ₄ ^2- ；GSSG；S ₈ ；CH ₃ SSSCH ₃ The method comprises the steps of carrying out a first treatment on the surface of the Cys; hcy; GSH), reactive oxygen species (ROS, including H ₂ O ₂ ；ClO ^- ；O ₂ ^- ；·OH； ^t BuOOH； ¹ O ₂ ) Reactive nitrogen (RNS, including NO; NO (NO) ₂ ^- ；ONOO ^- ；NO ₃ ^- ) Inorganic salt ion (K) ⁺ ；Ca ²⁺ ；Na ⁺ ；Mg ²⁺ ；Fe ³⁺ ；Zn ²⁺ ；Cu ²⁺ ；CO ₃ ^2- ；HCO ₃ ^- ；Cl ^- ；I ^- ；HPO ₄ ^2- ；H ₂ PO ₄ ^- ) Amino acids (Glu; trp; tyr; ser; arg; phe; his; ala; met; leu; val; pro; gly) will hardly generate chemiluminescent signals. Therefore, the probe CL-SN has the advantage of good selectivity to hydrogen polysulfide.

Example 4 influence of pH on the reaction of chemiluminescent Probe CL-SN with Hydrogen polysulfide and stability experiment of Probe CL-SN

(1) Effect of PH on the reaction of chemiluminescent Probe CL-SN with Hydrogen polysulfide

To not be provided withDetection of Na with pH vs. Probe CL-SN ₂ S ₄ Is to probe CL-SN and Na ₂ S ₄ Incubation was performed at different pH conditions. As shown in FIG. 7, the probe is relatively stable under different pH conditions and does not decompose. At Na (Na) ₂ S ₄ In the presence of the solution, when the pH value of the solution is 4.0 to 8.0, the concentration of phenoxy anions in the solution system is increased, and the chemiluminescence intensity is gradually enhanced. With the enhancement of the alkalinity of the solution, the enhancement of the protonation effect of the phenoxy anions gradually reduces the chemiluminescence intensity ^[8 2]. The greater intensity of chemiluminescence in the pH range of 7.0 to 9.0 suggests that probe CL-SN can sensitively detect hydrogen polysulfide under physiological conditions (ph=7.4).

(2) Stability test of Probe CL-SN

Probes were incubated in PBS (10 mM,10% FBS, pH 7.4) buffer for 7h at 37 ℃. As shown in the results of FIG. 8, although probe CL-SN produces a weak chemiluminescent signal in the buffer, it does not affect the detection of hydrogen polysulfide. The trace amount of probe CL-SN is decomposed because FBS in the system has esterase activity and reacts with the ester bond of the probe, thereby causing a weak response signal. Therefore, the probe can be used for detecting the hydrogen polysulfide sensitively even though being partially decomposed in a PBS buffer solution system containing 10% FBS, and has better stability.

Example 5 cell level luminescence imaging experiments

(1) Cell culture

HT-22 cells, H9c2 cells were inoculated in DMEM high-sugar medium containing 10% fetal bovine serum and 100. Mu.g/mL penicillin-streptomycin, and the cells were placed at 37℃with 5% CO ₂ Culturing in an incubator. When the cells grow to the logarithmic phase, the cells are passaged and inoculated in a black 96-well plate, and after the cells are grown by adherence, chemiluminescence imaging experiments at the cell level can be performed.

(2) Cytotoxic culture

The cytotoxicity of probe CL-SN was detected using the MTT method. HT-22 cells and H9c2 cells are inoculated on a 96-well cell culture plate with the density of 3-5 multiplied by 10 ⁴ And/or holes. After cell adhesion, the cell is adhered with probe CL-SN with different concentrations0,5,10,20,50. Mu.M) was incubated for 24h, then 20. Mu.L of MTT dye (3- [4, 5-dimethylthiazol-2-yl) was added to each well]-2, 5-diphenyltetrazolium bromide, 5mg/mL in PBS buffer), after 4h incubation the medium was removed, and 150 μl DMSO was added to dissolve formazan crystals, shaking up and absorbance at 570nm was measured using an microplate reader. Incubation was performed with no added probe as control wells and with medium added MTT as blank wells, at least three determinations for each sample. The calculation formula is as follows: cell relative viability = (experimental well OD value-blank well OD value)/(control well OD value-blank well OD value) ×100%. After further incubation with the probe CL-SN (10. Mu.M) for various times (0, 6,12,18,24 h) the procedure was repeated.

As shown in FIG. 10-A, when the concentration of probe CL-SN was less than 10. Mu.M, the cell viability was 90% or more, indicating that the probe CL-SN was very low in toxicity, and at a concentration of 10. Mu.M or less, there was no effect on the cell activity.

As shown in FIG. 10-B, the cell viability of the probe CL-SN (10. Mu.M) incubated with the cells for 0h,6h,12h,18h,24h was further examined, and the probe CL-SN incubated with the cells for 0-24h at a concentration of 10. Mu.M did not cause toxicity to the cells, and the viability reached more than 90%.

Example 6, cell level chemiluminescent imaging experiments

(1) Exogenous hydrogen polysulfide cell chemiluminescence imaging

The cells were divided into the following groups: (1) Probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) was incubated with H9c2 cells for 30min. (2) After incubation of probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) with H9c2 cells for 30min, different concentrations of Na were added ₂ S ₄ (25, 50. Mu.M, in 10. Mu.L of sample) for 10min. (3) H9c2 cells were pre-incubated with N-methylmaleimide (NMM, 1mM, in 10. Mu.L of saline) for 1H, then probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L of DMSO) was added for 30min, and Na was added ₂ S ₄ (50. Mu.M, in 10. Mu.L of sample) for 10min. Prior to imaging, the images were gently washed three times with phosphate buffer.

The experimental results are shown in FIG. 10, in which almost no chemiluminescent signal was observed after incubation of probe CL-SN with H9c2 cells for 30min (FIG. 10-A). After incubation of H9c2 cells with probe CL-SN for 30min, different concentrations were addedNa of (2) ₂ S ₄ After incubation (25. Mu.M, 50. Mu.M), the chemiluminescent intensity was significantly enhanced; and with addition of Na ₂ S ₄ The increase in concentration was further enhanced (675-fold, 1923-fold; FIG. 10-B; FIG. 10-C). After 1h pretreatment of cells with N-methylmaleimide (NMM as a hydrogen polysulfide scavenger), the cells were treated with probes CL-SN and Na ₂ S ₄ Incubation was followed with a significant decrease in chemiluminescent intensity (fig. 10D). The chemiluminescence in the figure is caused by exogenous hydrogen polysulfide, and the probe CL-SN can detect H9c2 cell exogenous hydrogen polysulfide.

(2) Cell chemiluminescence imaging of endogenous hydrogen polysulfide

The cells were divided into the following groups: (1) Probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L DMSO) was incubated with H9c2 cells for 30min. (2) After incubation of H9c2 cells with lipopolysaccharide (LPS, 1. Mu.g/mL, in 10. Mu.L of sample) for 16H, probe CL-SN (final concentration 10. Mu.M, in 1. Mu.L of DMSO) was added and incubated for 30min. (3) H9c2 cells were pre-incubated with DL-propargylglycine (PAG, 200. Mu.M, in 10. Mu.L of saline) for 30min, then lipopolysaccharide (LPS, 1. Mu.g/mL, in 10. Mu.L of saline) for 16H, and then incubated with probe CL-SN (final concentration 10. Mu.M, 1. Mu.L of DMSO) for 30min. Prior to imaging, the images were gently washed three times with phosphate buffer.

No chemiluminescent signal was observed after incubation of probe CL-SN with the cells (fig. 11-a). Cells incubated with LPS for 16h and then with probe CL-SN showed a significant chemiluminescent signal (2340-fold; FIG. 11-B). Subsequently, cells were pretreated with DL-propargylglycine (PAG, CSE enzyme inhibitor) and incubated with LPS, probes in sequence, with a significant decrease in chemiluminescent intensity (fig. 11-C). The result shows that the probe CL-SN can carry out chemiluminescence imaging detection on endogenous hydrogen polysulfide in cells.

(3) In vivo horizontal polysulfide hydrogen chemiluminescence imaging

3.1 animal feeding

Adult male mice of the healthy Kunming species, weighing 20-25g, were supplied by the laboratory animal center of the Xuzhou medical university (animal license number: SYXK (Su) 2007-0037). The animal protocol is approved by the animal protection and use committee of Xuzhou medical university, and the animal experiment is conducted in accordance with the rule of using experimental animals by Chinese law. Animals should be familiar with the environment a week in advance before the experiment, and are put into cage group culture under the natural circadian rhythm illumination condition, the temperature is 22+/-2 ℃, the humidity is 50+/-10%, and the animals can eat drinking water freely.

3.2 in vivo horizontal chemiluminescent imaging

Chemiluminescent imaging detection was performed using an LB983 NightOWL II small animal in vivo imager, and the bioluminescence pattern was selected for an exposure time of 60 seconds. Image and data analysis was performed using indiGo software.

Mice were divided into the following groups: (1) Mice were intraperitoneally injected with probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO's saline). (2) Abdominal injection of Na into mice ₂ S ₄ (2, 4mM, 100. Mu.L of sample) and then intraperitoneally injecting probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO of sample). (3) Mice were first intraperitoneally injected with LPS (10. Mu.g/mL, 100. Mu.L of saline) for 24h and then with probe CL-SN (100. Mu.M, 100. Mu.L, 10% DMSO of saline). (4) The mice were first intraperitoneally injected with DL-propargylglycine (2 mM, 100. Mu.L of saline) for 30min, then intraperitoneally injected with LPS (10. Mu.g/mL, 100. Mu.L of saline) for 24h, and then injected with probe CL-SN (100. Mu.M, 100. Mu.L of saline, 10% DMSO).

As shown in FIG. 12, the mice injected with the probe alone served as a control group, and had weak chemiluminescent signals on the abdomen (FIG. 12A). Compared with the control group, na with different concentration is injected into the abdominal cavity ₂ S ₄ After that, the chemiluminescent intensity increases significantly and with Na ₂ S ₄ The concentration was increased to increase (34-fold, 91-fold; fig. 12B, fig. 12C). This demonstrates that probe CL-SN can detect exogenous hydrogen polysulfide at different concentrations in mice. LPS-injected mice also showed a pronounced chemiluminescent signal on their abdomen (125-fold; FIG. 12-D). When mice were intraperitoneally injected with DL-Propargylglycine (PAG), the chemiluminescent intensity was significantly reduced after reinjecting the probe (fig. 12-E). This illustrates that the chemiluminescent signal in FIG. 11D is caused by LPS-induced endogenous hydrogen polysulfide. From this, probe CL-SN can detect endogenous hydrogen polysulfide in mice.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

1. A near infrared chemiluminescence probe of hydrogen polysulfide has a chemical structure shown in a formula (I),

2. the method for preparing the near infrared chemiluminescence probe of the hydrogen polysulfide, which is characterized by comprising the following synthetic route:

3. the method for preparing the near infrared luminescent probe for hydrogen polysulfide according to claim 2, characterized in that the reaction step comprises: the compound 1g and 2- (benzoylthio) benzoic acid are subjected to condensation reaction, and then the reaction product is catalyzed by methylene blue under the condition of introducing oxygen to obtain the compound of the formula (I).

4. The method for preparing a near infrared luminescent probe for hydrogen polysulfide according to claim 3, wherein 1g of the compound and 2- (benzoylthio) benzoic acid are dissolved in a solvent, EDC and DMAP are then added, and the reaction is stirred at room temperature for 1h; then under the condition of introducing oxygen and under the irradiation of yellow light, the compound of the formula (I) is obtained through the catalysis of methylene blue.

5. The method for preparing a near infrared luminescent probe for hydrogen polysulfide according to claim 4, wherein the molar ratio of 1g of compound to 2- (benzoylthio) benzoic acid in the reaction is 1:1 to 2, preferably 1:1.2 to 1.8; the solvent for the condensation reaction was methylene chloride.

6. The method for preparing the near infrared luminescence probe of hydrogen polysulfide according to claim 4, wherein the molar ratio of DMAP to EDC in the reaction is 1:2.8 to 3.2, preferably 1:3.0; the molar ratio of compound 1g to EDC was 1:0.5 to 2.5, preferably 1:1 to 2.

7. Use of the near infrared hydrogen polysulfide chemiluminescent probe of claim 1 for chemiluminescent imaging detection of endogenous hydrogen polysulfide in a cell or in a mouse.

8. The use of the near infrared chemiluminescent probe of claim 1 for quantitatively detecting hydrogen polysulfide.

9. Use of the near infrared chemiluminescent probe of hydrogen polysulfide of claim 1 for detecting the physiological and pathological mechanism of hydrogen polysulfide in an organism.

10. Use of the near infrared hydrogen polysulfide chemiluminescent probe of claim 1 for detecting the signal transduction pathway of hydrogen polysulfide in an organism.