Controllable visual carbon monoxide releasing agent, preparation method and application thereof

Abstract

The application discloses a controllable visual carbon monoxide releasing agent and a preparation method and application thereof, the releasing agent is Fla and Cys P molecules, Fla molecules are prepared by taking N,N-dimethyl-p-phenylenediamine as raw material, and the Fla molecules are prepared by cyclization, selenium dioxide oxidation, then hydroxy aldehyde condensation with 2-hydroxyacetophenone ketone under alkaline conditions, and esterification after acryloyl chloride; the structure of the Fla molecules is broken after light irradiation, and CO molecules are released; Cys P can react with cysteine (Cys) to generate flavone molecules Fla, and the releasing agent realizes visual detection of cysteine (Cys) and controllable release of CO.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical biology technology, and particularly relates to a controllable visual carbon monoxide releasing agent and a preparation method and application thereof. BACKGROUND

[0002] Carbon monoxide (CO) is a common gas, which is also ubiquitous in the organism and plays an important role in maintaining physiological functions. There are two pathways for the production of endogenous CO, one is lipid peroxidation, and the other is the process of generating hemin with hemin oxygenase. On the one hand, CO molecules can dilate blood vessels and have anti-inflammatory and anti-apoptotic effects, and on the other hand, the level of CO in the body is closely related to diseases such as atherosclerosis, vascular calcification, and pulmonary hypertension.

[0003] Therefore, how to efficiently deliver carbon monoxide in the organism to achieve the purpose of treatment has attracted widespread attention. However, direct inhalation of CO can easily cause carbon monoxide poisoning in patients, and the lack of targeting leads to blocked function. Therefore, developing a carrier with carbon monoxide transport and controllable release function has become the main research direction. At present, there are two main categories of molecules used as CO carriers: transition metal carbonyl complexes and small organic molecules. Metal carbonyl complexes are limited in application due to their inevitable metal toxicity, while small organic molecules do not have this limitation.

[0004] Common biological thiols in the human body include cysteine, homocysteine and glutathione, which play a crucial role in physiological and pathological processes. Cysteine (Cys) plays a role in maintaining redox homeostasis in the complex physiological environment of the human body. Too high or too low concentration of cysteine (Cys) can lead to abnormal physiological phenomena and the occurrence of diseases. For example, too high concentration of cysteine (Cys) can cause a series of nervous system diseases such as Alzheimer's disease; too low concentration of cysteine (Cys) can cause slow growth and development of humans, and inflammation in cells.

[0005] Therefore, as a marker of these abnormal physiological phenomena and diseases, it is crucial to rapidly and specifically test the cysteine (Cys) in the body for early clinical diagnosis and subsequent treatment. SUMMARY

[0006] To solve the above problems, the application discloses a controllable visual carbon monoxide releasing agent and a preparation method and application thereof, and provides a carbon monoxide releasing agent Fla and Cys P and a preparation method, wherein the Fla molecule is broken after light irradiation to release CO molecules; the Cys P can react with cysteine (Cys) to generate a flavone molecule Fla, and the application realizes visual detection of cysteine (Cys) and controllable release of CO, and provides a new reference tool for combination of clinical diagnosis and CO molecule treatment.

[0007] To achieve the above object, the technical scheme of the application is as follows:

[0008] A controllable visual carbon monoxide releasing agent comprises structures as shown in formula I and formula II.

[0009]

[0010] I II.

[0011] In the formula, the structure of formula I is Fla, and the structure of formula II is Cys P.

[0012] The preparation method of the controllable visual carbon monoxide releasing agent is characterized by the following specific steps.

[0013] (1) N,N-dimethyl-p-phenylenediamine is used as a raw material to synthesize N,N,2-trimethylquinolin-6-amine through cyclization;

[0014] (2) N,N,2-trimethylquinolin-6-amine obtained in step (1) is oxidized into 6-(dimethylamino)quinoline-2-carboxaldehyde through selenium dioxide oxidation;

[0015] (3) 6-(dimethylamino)quinoline-2-carboxaldehyde obtained in step (2) is subjected to aldol condensation with 2-hydroxyacetophenone under alkaline conditions to prepare 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla);

[0016] (4) 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla) obtained in step (3) is subjected to esterification reaction with acryloyl chloride to obtain 2-(6-(dimethylamino)quinolin-2-yl)-4-oxo-4H-chromen-3-yl acrylate (Cys P).

[0017] The reaction formula is as shown in the following formula:

[0018]

[0019] As an improvement of the application, the specific preparation steps of step (1) are as follows:

[0020] Step 1 : Dissolve N,N-dimethyl-p-phenylenediamine (36.7 mmol, 1 eq) in a solution of HCI (6 M), stir the mixture at room temperature for 1 h after addition of crotonaldehyde (73.5 mmol, 2 eq), then add toluene and reflux the reaction further overnight at 115 °C;

[0021] Step 2: After cooling to room temperature, remove the toluene layer and add a saturated solution of sodium hydroxide to neutralize the aqueous layer, extract the solution with dichloromethane, wash twice with a saturated solution of sodium chloride and dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure;

[0022] Step 3: Purify the crude N,N,2-trimethylquinolin-6-amine by column chromatography on silica gel (petroleum ether: ethyl acetate = 5: 1 v / v) to obtain N,N,2-trimethylquinolin-6-amine as a beige solid.

[0023] As an improvement of the present invention, the specific preparation steps of said step (2) are as follows:

[0024] Step 1 : Add selenium dioxide (25.1 mmol, 1.3 eq) to dioxane, heat the resulting mixture at 80 °C for 30 min, then add N,N,2-trimethylquinolin-6-amine (9.2 mmol, 1 eq) and stir at 80 °C for 10 h;

[0025] Step 2: After cooling to room temperature, filter the mixture through celite, then rinse the residue with small amounts of dichloromethane several times, concentrate the filtrate under reduced pressure;

[0026] Step 3: Purify the crude product obtained by column chromatography on silica gel (dichloromethane: methanol = 250: 1 v / v) to obtain 6-(dimethylamino)quinoline-2-carbaldehyde as a yellow solid.

[0027] As an improvement of the present invention, the specific preparation steps of said step (3) are as follows:

[0028] Step 1 : Add 6-(dimethylamino)quinoline-2-carbaldehyde (10 mmol) and 2- hydroxyacetophenone (10 mmol) to a round-bottom flask containing anhydrous methanol, then add sodium hydroxide (30 mmol) to the flask under stirring, then stir the mixture at room temperature for 24 hours, and successively add NaOH (10 mmol), 1 mL of water and 10 mL of hydrogen peroxide solution (30 %) under ice-water bath, finally stir at room temperature for 12 hours;

[0029] Step 2: Pour the mixture into ice water under stirring, add diluted sulfuric acid to adjust the pH value to 6, then extract with dichloromethane for three times, dry the dichloromethane with anhydrous sodium sulfate, filter, and finally concentrate in a rotary evaporator;

[0030] Step 3: Purify the obtained crude product by silica gel column chromatography (dichloromethane:methanol=100:1 v / v), and then further purify by recrystallization with dichloromethane to obtain 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla) as an orange solid.

[0031] As an improvement of the present application, the specific preparation steps of step (4) are as follows:

[0032] Step 1: Keep a mixture of 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla) (1 mmol) and triethylamine (7.2 mmol) in anhydrous dichloromethane for 30 minutes at -5 ℃, then slowly add acryloyl chloride (1.5 mmol) to the mixture, and react the reaction mixture at -5 ℃ for 8 h;

[0033] Step 2: Extract the mixture with dichloromethane, then wash the organic phase with water, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure;

[0034] Step 3: Purify the obtained crude product by column chromatography (dichloromethane:methanol=250:1 v / v) to obtain the pure product 2-(6-(dimethylamino)quinolin-2-yl)-4-oxo-4H-chromen-3-yl acrylate (Cys P) as an orange solid.

[0035] The present application provides an application of a controllable visual carbon monoxide releasing agent in releasing CO under light irradiation.

[0036] Wherein, the releasing agent Fla generates free radicals under light irradiation, then undergoes excited-state intramolecular proton transfer (ESIPT), then forms a triplet excited tautomer, and then reacts with oxygen to form a phenolic acid to release CO and decosyl.

[0037] The present application also provides an application of a controllable visual carbon monoxide releasing agent in detecting cysteine.

[0038] Wherein, the releasing agent Cys P uses flavone as a skeleton, and uses the thiol group and the amino group of cysteine (Cys) to perform nucleophilic addition and subsequent ring-closing reaction on the acryl double bond and the ester carbonyl of Cys P, respectively, to generate Fla.

[0039] The present application has the following beneficial effects:

[0040] 1. The carbon monoxide releasing agent Fla of the present application emits at 624 nm, which is longer than the similar backbone molecules reported in the current literature; exhibits good ratiometric fluorescence properties during the process of CO release under light irradiation; has a large Stokes shift, good anti-interference effect, small damage to biological samples, and strong sample penetration.

[0041] 2. The carbon monoxide releasing agent Cys P of the present application has good selectivity and can adapt to complex physiological environments; has high sensitivity, and the detection limit for cysteine (Cys) is 0.602 μM.

[0042] 3. The carbon monoxide releasing agent Cys P and Fla of the present application realize the visual detection of the driving factor Cys and the controllable release of CO, and provide a new reference tool for the combination of clinical diagnosis and CO molecular therapy. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 is a fluorescence spectrum of Fla without light irradiation; Fig. 2 is a fluorescence spectrum of Fla without the addition of GSH under light irradiation; Fig. 3 is a fluorescence spectrum of Fla after the addition of GSH under light irradiation; and Fig. 4 is a ratiometric fluorescence fitting curve diagram.

[0044] Figure 2 Fig. 5 is an ultraviolet-visible absorption curve of Fla before and after light irradiation and a gas chromatogram at different light irradiation times.

[0045] Figure 3 Fig. 6 is an excitation spectrum, an emission spectrum, and an ultraviolet-visible absorption spectrum of Cys P itself and after the addition of Cys.

[0046] Figure 4 Fig. 7 is a fluorescence spectrum intensity change diagram and a fitting curve diagram of Cys P under in vitro test conditions, after the addition of 10 times the amount of Cys and incubation for different times.

[0047] Figure 5 Fig. 8 is a fluorescence spectrum intensity change diagram and a fitting curve diagram of Cys P under in vitro test conditions, after the addition of Cys with different concentrations.

[0048] Figure 6 Fig. 9 is a fluorescence spectrum intensity change diagram and a linear relationship curve diagram of Cys P under in vitro test conditions, after the addition of 0-50 μM Cys.

[0049] Figure 7 Fig. 10 is a fluorescence spectrum dependence curve diagram of Cys P under different pH conditions before and after the addition of Cys.

[0050] Figure 8 Fig. 11 is a fluorescence spectrum intensity change diagram and a fluorescence intensity columnar diagram of Cys P under in vitro test conditions, after the addition of different metal cations.

[0051] Figure 9 This is a schematic diagram and a fluorescence intensity bar chart showing the changes in fluorescence intensity of Cys P under in vitro testing conditions with the addition of different amino acids and biothiols.

[0052] Figure 10 This is a schematic diagram and a fluorescence intensity bar chart showing the changes in fluorescence intensity of Cys P under in vitro testing conditions with the addition of different anions.

[0053] Figure 11 This is a bar chart showing the interference fluorescence intensity of Cys P under in vitro testing conditions with the addition of different ions, amino acids, and biothiols.

[0054] Figure 12 TLC and high-performance liquid chromatography chromatograms of the Cys P addition reaction of this invention.

[0055] Figure 13 Fla obtained in Example 1 1 H NMR spectrum.

[0056] Figure 14 Fla obtained in Example 1 13 C10 NMR spectrum.

[0057] Figure 15 The mass spectrum (HR-MS) of Fla obtained in Example 1.

[0058] Figure 16 Cys P prepared in Example 2 1 H NMR spectrum.

[0059] Figure 17 Cys P prepared in Example 2 13 C10 NMR spectrum.

[0060] Figure 18 The mass spectrum (HR-MS) of Cys P obtained in Example 2. Detailed Implementation

[0061] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0062] Example 1: Synthesis of the carbon monoxide release agent molecule Fla.

[0063] (1) N,N-dimethyl-p-phenylenediamine (36.7 mmol, 1.0 eq) was dissolved in a HC1 (6 M) solution, after the addition of crotonaldehyde (73.5 mmol, 2 eq), the mixture was stirred at room temperature for 1 h. Then toluene was added and the reaction was further refluxed at 115 °C overnight. After cooling to room temperature, the toluene layer was removed and a saturated sodium hydroxide solution was added to neutralize the aqueous layer. The solution was extracted with dichloromethane, washed twice with a saturated sodium chloride solution and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude N,N,2-trimethylquinolin-6-amine was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1 v / v) to give N,N,2-trimethylquinolin-6-amine as a sandy brown solid;

[0064] (2) Selenium dioxide (25.1 mmol, 1.3 eq) was added to dioxane, the resulting mixture was heated at 80 °C for 30 min, then N,N,2-trimethylquinolin-6-amine (9.2 mmol, 1 eq) was added and stirred at 80 °C for 10 h. After cooling to room temperature, the mixture was filtered through celite, then the filter residue was rinsed several times with a small amount of dichloromethane. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 250:1 v / v) to give 6-(dimethylamino)quinoline-2-carbaldehyde as a yellow solid;

[0065] (3) 6-(dimethylamino)quinoline-2-carbaldehyde (10 mmol) and 2-hydroxyacetophenone (10 mmol) were added to a round bottom flask containing anhydrous methanol. Then, sodium hydroxide (30 mmol) was added to the flask under stirring. The mixture was stirred at room temperature for 24 hours, and NaOH (10 mmol), 1 mL of water and 10 mL of hydrogen peroxide solution (30 %) were added successively under ice water bath. Then, stirring was continued at room temperature for 12 hours. The mixture was poured into ice water under stirring, dilute sulfuric acid was added to adjust the pH value to 6. It was extracted with dichloromethane three times. Then the dichloromethane was dried over anhydrous sodium sulfate, filtered, and concentrated in a rotary evaporator. The obtained crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100:1 v / v). Subsequently, further purification by recrystallization from dichloromethane gave 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla) as an orange solid; the structure of the obtained compound Fla was fully characterized by 1 H NMR ( Figure 13 ) and 13 C NMR ( Figure 14 ) spectra and mass spectrometry (HR-MS) Figure 15 ).

[0066] 1H NMR (400 MHz, Chloroform-d) δ 13.98 (s, 1H), 8.36 (dd, J = 8.0,1.6 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H), 7.88 (d, J =9.3 Hz, 1H), 7.69 (ddd, J = 8.6, 7.0, 1.6 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H),7.44 – 7.35 (m, 2H), 6.80 (d, J = 2.7 Hz, 1H), 3.13 (s,6H). 13 C NMR (101 MHz,Chloroform-d) δ 154.50, 149.30, 148.03, 143.34, 138.54, 135.59, 133.12,128.61, 126.10, 124.08, 120.27, 117.83, 117.22, 104.21, 77.23, 40.45.ESI: m / z c 20 H 16 N2O3, 332.1161 [M+H] + , found 333.1240. 。

[0067] Example 2: Synthesis of carbon monoxide releasing agent molecule Cys P.

[0068] A mixture of 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromen-4-one (Fla) synthesized in example 1 (1 mmol) and triethylamine (7.2 mmol) in anhydrous dichloromethane was kept at -5 °C for 30 min, then acryloyl chloride (1.5 mmol) was slowly added to the mixture. The reaction mixture was reacted at -5 °C for 8 h. The mixture was extracted with dichloromethane, then the organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by column chromatography (dichloromethane:methanol = 250:1 v / v) to obtain the pure product 2-(6-(dimethylamino)quinolin-2-yl)-4-oxo-4H-chromen-3-yl acrylate (Cys P) as an orange solid. The structure of the obtained compound Cys P was confirmed by 1 H NMR ( Figure 16) and 13 C NMR ( Figure 17 ) spectra and mass spectrometry (HR-MS) ( Figure 18 ) fully characterized.

[0069] 1 H NMR (400 MHz, Chloroform-d) δ 8.32 (dd, J = 8.0, 1.7 Hz, 1H), 8.05 (dd, J = 8.7, 0.8 Hz, 1H), 7.95 - 7.89 (m, 2H), 7.75 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.66 (dd, J = 8.6, 1.1 Hz, 1H), 7.47 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.39 (dd, J = 9.4, 2.8 Hz, 1H), 6.81 - 6.66 (m, 2H), 6.52 (dd, J = 17.4, 10.4 Hz, 1H), 6.11 (dd, J = 10.4, 1.4 Hz, 1H), 3.15 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 172.91, 163.81, 155.49, 149.61, 143.93, 141.86, 133.98, 133.92, 132.96, 131.09, 130.10, 127.71, 126.17, 125.19, 123.93, 120.22, 119.76, 118.30, 103.74, 77.25, 40.49. ESI: m / z cald. For C 23 H 18 N2O4, 386.1267 [M+H] + , found 387.1341..

[0070] Example 3: Final structure of carbon monoxide releasing agent molecule Fla was determined by 1 H and 13 C NMR spectra and high resolution mass spectrometry fully confirmed.

[0071] The carbon monoxide releasing molecule Fla prepared in Example 1 was dissolved in DMSO to make 1 mM and 500 μM stock solutions, and all test results were obtained from three or more parallel experiments.

[0072] Fla photoluminescence test: 100 μL Fla, 900 μL PBS buffer (pH 7.4) were mixed to make 1 mL test solution, which was irradiated by a white LED lamp (36 w, 3240 LM) at 25 °C, and the fluorescence intensity was recorded every 2 minutes for a total of 20 minutes. The fluorescence intensity change was tested by a fluorescence spectrometer. Ex = 420 nm, slit = 5 nm, and the test was divided into three groups: one group was not irradiated and placed in the dark; one group was tested after irradiation; and the other group was tested after adding GSH (1 mM) and irradiation. The test results are shown in Figure 1 By fitting the ratio fluorescence curve, horizontally, the peak intensity at 624 nm continuously decreased and the peak intensity at 535 nm continuously increased with the increase of irradiation time, showing good ratio fluorescence properties. Vertically, the fluorescence curve of Fla did not change without irradiation. After adding GSH, the fluorescence curve changed more obviously than without adding. This means that Fla undergoes structural changes under irradiation, and high concentrations of GSH in the physiological environment can promote the change process.

[0073] Fla photoluminescence ultraviolet-visible and gas chromatography test: 100 μL Fla, 900 μL PBS buffer (pH 7.4) were mixed to make 1 mL test solution, which was injected into a 2 mL mass spectrometer bottle filled with argon and sealed. After irradiation for 20 minutes at 25 °C using a white LED lamp (36 w, 3240 LM), the liquid phase change was detected by an ultraviolet spectrometer, and the gas change in the bottle was detected by a gas chromatograph. As shown in Figure 2 The ultraviolet results show that the Fla absorption peak at 418 nm is significantly reduced after irradiation. At the same time, on the gas chromatogram, the CO peak intensity continuously increases with the increase of irradiation time, indicating that carbon monoxide is released during irradiation. Therefore, combined with the above fluorescence, ultraviolet and gas chromatography results, it is confirmed that Fla releases gaseous carbon monoxide under irradiation.

[0074] Example 4: Final structure of carbon monoxide releasing molecule Cys P 1 H and 13 C NMR spectrum and high resolution mass spectrum completely confirm the preliminary in vitro test with PBS buffer.

[0075] The carbon monoxide releasing and molecular Cys P prepared in Example 2 was formulated into 1 mM and 500 μM stock solutions with DMSO, and cysteine (Cys) was formulated into 1 mM stock solution with distilled water. The final concentration of Cys P for testing was 5 μM, and all test results were obtained from three or more parallel experiments.

[0076] 10 μL of Cys P was taken and mixed with 990 μL of PBS buffer (pH 7.4) to prepare 1 mL of the test solution. Then 10 μL of Cys P was taken and mixed with 100 μL of Cys and 890 μL of PBS buffer (pH 7.4) to prepare 1 mL of the test solution. After incubation at 37 °C for 5 min, the UV absorption and fluorescence spectra of Cys P before and after response were measured, as shown in Figure 3 The results show that Cys P responds rapidly to cysteine (Cys), and the maximum emission wavelength after response is Em=624 nm, Ex=420 nm. At the same time, the UV absorption shows that the absorption of Cys P before response is 437 nm, and the UV absorption after response is 418 nm, which is consistent with the absorption curve of Fla.

[0077] Time kinetics test of Cys P: 10 μL of Cys P was taken and mixed with 100 μL of Cys and 890 μL of PBS buffer (pH 7.4) to prepare 1 mL of the test solution. After shaking uniformly, the fluorescence intensity change was immediately tested by a fluorescence spectrometer. Ex=420 nm, slit width was 5 nm. The test results are shown in Figure 4 Through nonlinear fitting of the fluorescence intensity, we determined the subsequent incubation time as 15 min according to Figure 4 .

[0078] Cys P concentration gradient test for cysteine (Cys): 10 μL of Cys P was taken and mixed with 100 μL of Cys and 890 μL of PBS buffer (pH 7.4) to prepare 1 mL of the test solution. After incubation at 37 °C for 15 min, the fluorescence intensity change was immediately tested by a fluorescence spectrometer. Ex=420 nm, slit width was 5 nm, and the final concentration of Cys was 0-200 μM. The test results are shown in Figure 5 and Figure 6 We linearly fitted the relationship between concentration and fluorescence intensity (R 2 =0.9874), and calculated the detection limit LOD=0.602 μM based on the background noise, indicating that Cys P exhibits high sensitivity for cysteine (Cys) detection.

[0079] Cys P for different pH conditions on the fluorescence spectrum dependence test: take 10 μL Cys P, 990 μL PBS buffer (pH 7.4) into 1 mL of the test solution. Again take 10 μL Cys P, 100 μL Cys, 890 μL PBS buffer (pH 7.4) into 1 mL of the test solution, incubated at 37 ℃ for 15 min. Shake immediately after uniform with fluorescence spectrometer test its fluorescence intensity change. Ex = 420 nm, slit for 5 nm, PBS buffer solution pH range: 5-11. Test results as shown in Figure 7 The results show that Cys P in the pH conditions of 6-10, has a high stability, so it will have good adaptability in the physiological environment.

[0080] Cys P of the selective test: take 10 μL Cys P, 100 μL interfering ions, 890 μL PBS buffer (pH 7.4) into 1 mL of the test solution, incubated at 37 ℃ for 15 min. Shake immediately after uniform with fluorescence spectrometer test its fluorescence intensity change. Ex = 420 nm, slit for 5 nm, metal ions include: Na + , K + , Mg 2+ , Ca 2+ , Ba 2+ , Al 3+ , Pb 2+ , Cr 3+ , Mn 2+ , Fe 2+ , Fe 3+ , Sn 2+ , Ni 2+ , Cu 2+ , Cu + , Zn 2+ , Cd 2+ , Ag + . Amino acids include: Hcy, GSH, Lys, Arg, Ala, Thr, Met, Asn, Ile, Asp, Val, Ser, Pro, Glu, Leu, Gly, His, Tyr, Phe. Anions include: S 2− , HS − , SCN − , HSO3 − , SO3 2− , SO4 2− , CO3 2− , HCO3 − , NO3 −, NO2 − , AcO − , Br − , Cl − , F − . The test results are shown in Table 10. In the test of a large number of ions and amino acids, Cys P showed high selectivity and specifically reacted with Cys. Figure 8 , 9 ,10. In the test of a large number of ions and amino acids, Cys P showed high selectivity and specifically reacted with Cys.

[0081] Interference test of Cys P: 10 μL of Cys P, 100 μL of interfering ions, 890 μL of PBS buffer (pH 7.4) were mixed to make 1 mL of test solution, and incubated at 37 ℃ for 15 min. After shaking, the fluorescence intensity change was immediately tested by fluorescence spectrometer. Ex = 420 nm, slit = 5 nm, and multiple test groups were configured. Among them, there were anion mixed groups: S 2− , HS − , SCN − , HSO3 − , SO3 2− , SO4 2− , CO3 2− , HCO3 − , NO3 − , NO2 − , AcO − , Br − , Cl − , F − ; amino acid mixed groups: Hcy, GSH, Lys, Arg, Ala, Thr, Met, Asn, Ile, Asp, Val, Ser, Pro, Glu, Leu, Gly, His, Tyr, Phe; metal ion mixed groups: Na + , K + , Mg 2+ , Ca 2+ , Ba 2+ , Al 3+ , Pb 2+ , Cr 3+ , Mn 2 + , Fe 2+ , Fe 3+ , Sn 2+ , Ni 2+ , Cu 2+ , Cu + , Zn 2+ , Cd 2+ , Ag+ The test results are as follows: Figure 11 As shown in the figure, the results indicate that even in a mixture of various ions and amino acids, Cys P still exhibits strong selectivity and sensitivity to cysteine, demonstrating its strong anti-interference ability. In fact, this simulates a simple physiological environment, which has high reference value for the application of Cys P in physiological systems.

[0082] TLC and HPLC experiments of Cys P: Results are as follows Figure 12 As shown in the TLC images, A represents Cys P, C represents Fla, and B represents the extract of Cys P after incubation with Cys for 15 minutes under in vitro testing conditions. The TLC results indicate that the product responding to Cys P exhibits Fla polarity. In the high-performance liquid chromatography (HPLC) experiments, isocratic elution was used with a mixture of acetonitrile and water (v / v, 7:3). The retention times of Cys P, Cys P + Cys, and Fla under in vitro testing conditions were obtained. The results show that with increasing incubation time, the peak area and height of Cys P continuously decrease, while the peak area and height of Fla, as the product peak, continuously increase. Combined with TLC, Figure 1 The fluorescence and UV results shown indicate that cysteine ​​(Cys), as a triggering factor, can specifically, rapidly, and efficiently activate the acrylate switch of Cys P in an in vitro testing environment, releasing the flavonoid Fla. Furthermore, in this process, Cys P enables the visual detection of cysteine ​​(Cys).

[0083] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made on the basis of the above embodiments without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A controllable and visible carbon monoxide release agent, characterized in that: The carbon monoxide releasing agent comprises the structures shown in Formula I and Formula II: ； Formula I has a structure of Fla, and Formula II has a structure of Cys P.

2. A method for preparing the controllable and visualized carbon monoxide releasing agent according to claim 1, characterized in that, The specific steps are as follows: (1) Using N,N-dimethyl-p-phenylenediamine as a raw material, N,N,2-trimethylquinoline-6-amine was synthesized by cyclization; (2) The N,N,2-trimethylquinoline-6-amine obtained in step (1) is oxidized to 6-(dimethylamino)quinoline-2-carboxaldehyde by selenium dioxide; (3) The 6-(dimethylamino)quinoline-2-carboxaldehyde obtained in step (2) was subjected to aldol condensation with 2-hydroxyacetophenone under alkaline conditions to prepare 2-(6-(dimethylamino)quinoline-2-yl)-3-hydroxy-4H-chromone-4-one (Fla). (4) The 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromone-4-one (Fla) obtained in step (3) is subjected to esterification reaction with acryloyl chloride to obtain 2-(6-(dimethylamino)quinolin-2-yl)-4-oxo-4H-chromone-3-yl acrylate (Cys P).

3. The method for preparing a controllable and visualized carbon monoxide releasing agent according to claim 2, characterized in that, The specific preparation steps of step (1) are as follows: Step 1: Dissolve 36.7 mmol, 1 equivalent of N,N-dimethyl-p-phenylenediamine in 6 M HCl solution, add 73.5 mmol, 2 equivalent of crotonaldehyde, stir the mixture at room temperature for 1 h, then add toluene, and reflux at 115 °C overnight. Step 2: After cooling to room temperature, remove the toluene layer, add saturated sodium hydroxide solution to neutralize the aqueous layer, extract the solution with dichloromethane, wash twice with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure; Step 3: The crude N,N,2-trimethylquinoline-6-amine product was purified by silica gel column chromatography to obtain a sandy brown solid N,N,2-trimethylquinoline-6-amine. The eluent for the silica gel column chromatography was petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 5:

1.

4. The method for preparing a controllable and visualized carbon monoxide releasing agent according to claim 2, characterized in that, The specific preparation steps of step (2) are as follows: Step 1: 25.1 mmol (1.3 equivalents) of selenium dioxide was added to dioxane, the resulting mixture was heated at 80 °C for 30 min, and then 9.2 mmol (1 equivalent) of N,N,2-trimethylquinoline-6-amine was added and stirred at 80 °C for 10 h. Step 2: After cooling to room temperature, filter the mixture through diatomaceous earth, then wash the filter residue several times with a small amount of dichloromethane, and concentrate the filtrate under reduced pressure. Step 3: The crude product was purified by silica gel column chromatography to obtain 6-(dimethylamino)quinoline-2-carboxaldehyde in the form of a yellow solid. The eluent for the silica gel column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 250:

1.

5. The method for preparing a controllable and visualized carbon monoxide releasing agent according to claim 2, characterized in that, The specific preparation steps of step (3) are as follows: Step 1: Add 10 mmol of 6-(dimethylamino)quinoline-2-carboxaldehyde and 10 mmol of 2-hydroxyacetophenone to a round-bottom flask containing anhydrous methanol, then add 30 mmol of sodium hydroxide to the flask with stirring. The mixture is then stirred at room temperature for 24 hours, and 10 mmol of NaOH, 1 mL of water and 10 mL of 30% hydrogen peroxide solution are added sequentially in an ice-water bath. Finally, the mixture is stirred at room temperature for 12 hours. Step 2: Pour the mixture into ice water with stirring, add diluted sulfuric acid to adjust the pH to 6, then extract three times with dichloromethane, dry the dichloromethane with anhydrous sodium sulfate, filter, and finally concentrate in a rotary evaporator. Step 3: The crude product obtained was purified by silica gel column chromatography, and then further purified by recrystallization from dichloromethane to obtain an orange solid 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromone-4-one (Fla). The eluent for the silica gel column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 100:

1.

6. The method for preparing a controllable and visualized carbon monoxide releasing agent according to claim 2, characterized in that, The specific preparation steps of step (4) are as follows: Step 1: A mixture of 1 mmol 2-(6-(dimethylamino)quinolin-2-yl)-3-hydroxy-4H-chromone-4-one (Fla) and 7.2 mmol triethylamine in anhydrous dichloromethane was kept at -5 °C for 30 min, and then 1.5 mmol acryloyl chloride was slowly added to the mixture. The reaction mixture was reacted at -5 °C for 8 h. Step 2: Extract the mixture with dichloromethane, then wash the organic phase with water, dry it on anhydrous sodium sulfate, filter it, and concentrate the filtrate under reduced pressure; Step 3: The crude product was obtained by column chromatography purification to obtain the orange solid pure product 2-(6-(dimethylamino)quinoline-2-yl)-4-oxo-4H-chromone-3-yl acrylate (Cys P). The column chromatography eluent was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 250:

1.

7. The application of the controllable and visualized carbon monoxide release agent according to claim 1 in CO release under light.

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