A method for detecting endogenous H 2 S-containing copper ion biological fluorescent probe and its preparation method and application

Abstract

The invention discloses a method for detecting endogenous H 2 A copper ion-containing biological fluorescent probe of S and its preparation method and application. It belongs to the field of fluorescence detection technology. The probe structural formula is #imgabs0#. The probe is effective for endogenous H 2 S has high sensitivity, low detection limit and excellent selectivity. It also has low toxicity, high biosafety and good water solubility, and is suitable for endogenous H 2 In addition, it can detect endogenous H 2 S has a short response time and can quickly and accurately monitor intracellular H 2 S level, and has potential application value in the research field of related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and relates to a copper ion-containing biological fluorescent probe for detecting endogenous H2S in cells, and a preparation method and application thereof. Background Art

[0002] Endogenous hydrogen sulfide (H2S) is synthesized by mammalian tissues through two pyridoxal-5′-phosphate-dependent enzymes responsible for the metabolism of L-cysteine, namely, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), and through a third pathway involving the combined action of 3-mercaptopyruvate sulfurtransferase (3-MST) and cysteine ​​aminotransferase (CAT). Several studies have shown that endogenous H2S production and exogenous H2S administration can affect a variety of pathologies. As an important endogenous gaseous signaling compound, H2S plays a vital role in cellular physiology and pathology and is considered the third most important gaseous transmitter in the regulation of cardiovascular, neuronal, immune, endocrine, and gastrointestinal systems, alongside nitric oxide (NO) and carbon monoxide (CO). In addition, endogenous H2S acts as a signaling molecule to regulate blood pressure and neuronal activity, relax vascular smooth muscle, inhibit insulin signaling, and has anti-inflammatory and anti-apoptotic properties. Multiple disease phenotypes are associated with endogenous H2S levels, such as Alzheimer's disease, Down syndrome, diabetes, and cirrhosis. Therefore, the detection of endogenous H2S is crucial for the study of biological and pathological processes in vivo.

[0003] Many methods for analyzing and detecting H2S have been developed in the prior art, such as electrochemical method, gas chromatography, liquid chromatography, colorimetry, methylene blue analysis, etc. However, these methods have the disadvantages of complex operation, expensive instruments, poor selectivity, and even certain destructiveness to cells, which greatly limits their practical application. In contrast, fluorescent probes not only have high selectivity, high sensitivity, and timely imaging, but also are simple to operate and low in cost, so they have received extensive attention in the qualitative and quantitative detection of H2S. For example, Chinese patent application with publication number CN117024395A discloses a flavonol derivative fluorescent probe for detecting H2S, and its preparation method and application; Chinese patent application with publication number CN116063301A discloses a benzothiazole derivative fluorescent probe, and its preparation method and application; Chinese patent application with publication number CN107501221A discloses a fluorescent probe that responds rapidly to hydrogen sulfide in vivo, and its preparation method and application; however, these fluorescent probes have poor sensitivity and accuracy, and as the test proceeds, problems such as redox homeostasis imbalance caused by intracellular hydrogen sulfide consumption limit their application in the field of endogenous hydrogen sulfide detection in organisms. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a copper ion-containing biological fluorescent probe for detecting endogenous H2S in cells, and a preparation method and application thereof, thereby solving the technical problems that the existing fluorescent probe has a small Stokes shift, which affects the sensitivity and accuracy of detection and affects the redox homeostasis in cells.

[0005] The present invention is achieved through the following technical solutions:

[0006] A copper ion-containing biological fluorescent probe for detecting endogenous H2S in cells, the structural formula of the fluorescent probe is as follows:

[0007] .

[0008] Preferably, the above-mentioned method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells comprises the following steps:

[0009] S1: adding a chloroform solution of benzyl chloroformate dropwise to a chloroform solution of 1,4,7,10-tetraazacyclododecane, and reacting at room temperature to obtain intermediate 1;

[0010] The structural formula of the intermediate 1 is:

[0011] S2: adding triethylamine and tert-butyl bromoacetate in acetonitrile to the acetonitrile solution of the intermediate 1 in sequence, and refluxing and stirring to react to obtain the intermediate 2;

[0012] The structural formula of the intermediate 2 is:

[0013] S3: adding a catalyst to the methanol solution of the intermediate 2, stirring and reacting under a hydrogen atmosphere to obtain an intermediate 3;

[0014] The structural formula of the intermediate 3 is:

[0015] S4: adding potassium carbonate to the chloroform solution of the intermediate 3, and adding an organic solution of dansyl chloride, and after the reaction is completed, the intermediate 4 is obtained;

[0016] The structural formula of the intermediate 4 is:

[0017] S5: adding the intermediate 4 to a mixed solution of dichloromethane and trifluoroacetic acid, stirring for reaction, and obtaining a fluorescent probe ligand L;

[0018] The structural formula of the fluorescent probe ligand L is:

[0019] S6: dissolving the fluorescent probe ligand L in THF, adjusting the pH value of the solution to 7-8, then adding copper chloride and stirring the reaction to obtain the copper ion-containing biological fluorescent probe for detecting endogenous H2S in cells.

[0020] Preferably, in step S1, the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is (2-2.2):1.

[0021] Preferably, the reaction temperature in step S2 is 60-90° C., and the reaction time is 42-48 h.

[0022] Preferably, in step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and bromic acid / acetic acid. For the palladium-carbon catalyst, the mass fraction of palladium to carbon is 5% to 10%, and for the palladium hydroxide-carbon catalyst, the mass fraction of palladium hydroxide to carbon is 10% to 20%.

[0023] Preferably, in step S4, the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:(1.6-1.8):(2.2-2.4).

[0024] Preferably, in step S5, the reaction time is 12 to 16 h.

[0025] Preferably, in step S6, the reaction temperature is 60-65° C., the reaction time is 4-5 h; and the molar ratio of the probe ligand L to copper chloride is 1:(1.1-1.3).

[0026] The copper ion-containing bioluminescent probe is used in the preparation of a kit for detecting endogenous H2S in cells.

[0027] A kit for detecting endogenous H2S in cells comprises the above-mentioned copper ion-containing bioluminescent probe for detecting endogenous H2S in cells.

[0028] Compared with the prior art, the present invention has the following beneficial technical effects:

[0029] The present invention discloses a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells. The probe is an N-heterocyclic fluorescent probe chelated with copper ions. The nitrogen atom of the N-heterocyclic (i.e., ligand L) is grafted with carboxymethyl and dansyl groups. Firstly, the dansyl group itself has an obvious fluorescence effect. 2+ It has significant paramagnetism. The ligand L reacts with Cu 2+ When hydrogen sulfide is present in the system, S 2- Can be used with Cu 2+ The reaction forms copper sulfide precipitate, which is released from the azole macrocycle, that is, the macrocycle of ligand L releases Cu 2+ , so that the fluorescence characteristics of ligand L are restored. Therefore, the detection of hydrogen sulfide is effectively achieved through the recovery process of probe fluorescence. In addition, carboxymethyl and dansyl groups are grafted on the nitrogen atom of its N heterocycle (i.e., ligand L), and the oxygen atom on the carboxymethyl group promotes the chelation between ligand L and copper ions due to its obvious lone pair of electrons, effectively improving the sensitivity of detection. At the same time, the copper sulfide generated in the detection process of the present invention is a reversible process, which has little damage to the intracellular redox homeostasis, has a short response time, and can quickly and accurately detect hydrogen sulfide in real time.

[0030] In addition, the present invention also discloses a method for preparing the copper ion-containing bioluminescent probe for detecting endogenous H2S in cells. In the preparation process, cyclopentane (Cyclen), i.e., 1,4,7,10-tetraazacyclododecane, is used as the initial reactant, and the tert-butyl carbonyl group on its derivative 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane (DO2A, i.e., intermediate 3) is subsequently converted into a carboxyl group by acidification, and the carboxyl group effectively enhances the chelating ability and water solubility of the compound, especially in the in vivo environment. The remaining secondary amine on DO2A is covalently coupled with dansyl chloride (Cl-DNS) to obtain 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane, i.e., probe ligand L. Finally, the probe ligand L reacts with copper chloride to obtain the fluorescent probe Cu-L. At the same time, in the early stage of preparation, the chloroformyl solution of benzyl chloroformate is added dropwise to the chloroformyl solution of 1,4,7,10-tetraazacyclododecane to avoid the formation of by-products and improve the purity and yield of the product. The method has a simple process and a reasonable design. It effectively realizes the preparation of ligands and the effective chelation of copper ions, and successfully prepares high-purity products. The probe is sensitive to the microenvironment, the system preparation is simple, the chemical stability is good, and the selectivity is good. It has high sensitivity, low detection limit and excellent selectivity for biological thiols, anions and amino acids. It also has low toxicity, high biosafety, and is suitable for intracellular H2S imaging. It can monitor intracellular H2S quickly, accurately and specifically.

[0031] Furthermore, in step S1, the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is (2-2.2):1, and the molar ratio of the reactants is controlled to control the combination of the benzyloxycarbonyl protecting group and the amino group to form carbamate to protect the amino group, thereby obtaining a para-protected reaction intermediate.

[0032] Furthermore, the reaction temperature in step S2 is 60-90° C. and the reaction time is 42-48 h, which allows tert-butyl bromoacetate to fully combine with the remaining two amino groups on the macrocycle to form a para-type linker arm.

[0033] Further, in step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and bromic acid / acetic acid. For the palladium-carbon catalyst, the mass fraction of palladium to carbon is 5% to 10%, and for the palladium hydroxide-carbon catalyst, the mass fraction of palladium hydroxide to carbon is 10% to 20%, which can deprotect the amino group protected by the benzyloxycarbonyl group and reform the reaction site.

[0034] Furthermore, in step S4, the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:(1.6-1.8):(2.2-2.4), so that dansyl chloride reacts with amino group under alkaline conditions to form an intermediate with dansyl group substituted at the para position.

[0035] Furthermore, in step S5, the reaction time is 12 to 16 h, which allows the tert-butyl ester group to be fully hydrolyzed into a carboxylic acid group under acidic conditions, thereby providing a coordination site for the next step of complexing metal ions.

[0036] Further, in step S6, the reaction temperature is 60-65°C, the reaction time is 4-5 h; and the molar ratio of the probe ligand L to copper chloride is 1:(1.1-1.3), so that Cu 2+ It forms a six-coordinated complex structure with the macrocyclic ring of the dicarboxyl linker arm. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 A synthetic route of a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells in the present invention;

[0039] Figure 2 The probe Cu-L (10 μM) prepared in Example 1 of the present invention was tested at different equivalents of Cu 2+ Fluorescence emission spectra of Cu-L (0~1eq) (A), and the fluorescence intensity of the probe Cu-L and Cu 2+ Correlation curve between equivalents (B);

[0040] Figure 3 The probe Cu-L (10 μM) prepared in Example 1 of the present invention is in the range of 0-80 μM S 2- Fluorescence emission spectra of the probe Cu-L at λex = 340 nm (A) and the fluorescence intensity of the probe Cu-L versus S 2- Correlation curve between concentrations (B);

[0041] Figure 4 The fluorescence response of the probe Cu-L (10 µM) prepared in Example 1 of the present invention to sodium sulfide (60 µM) changes over time (0-5 min) (λex = 340 nm);

[0042] Figure 5The probe Cu-L (10 μM) prepared in Example 1 of the present invention and S 2- (60µM) with other sulfur-free anions Ac - 、NO3 - , Cl - 、CO3 2- 、HCO3 - , HCO - (60µM) fluorescence intensity comparison, all tests were performed using PBS solution;

[0043] Figure 6 The probe Cu-L (10 μM) prepared in Example 1 of the present invention and 60 μM S 2- and SO4 2- 、SO3 2- 、HSO3 - 、S2O8 2- 、S2O3 2- and HS - Comparison of fluorescence intensity, all tests were performed using PBS solution;

[0044] Figure 7 The probe Cu-L (10 μM) prepared in Example 1 of the present invention and 60 μM S 2- , amino acids, and biothiols (Thr, Gly, Tyr, Trp, Leu, Glu, Lys, Cys, Hcy, GSH) fluorescence intensity comparison, all tests were performed using PBS solution;

[0045] Figure 8 The cytotoxicity test results of the probe Cu-L prepared in Example 1 of the present invention in 0-50 μM culture medium;

[0046] Fig. 9 In order to add S to the probe Cu-L prepared in Example 1 of the present invention 2- Comparison of confocal fluorescence intensity before and after ionization.

[0047] Fig.10 In order to add S to the probe Cu-L prepared in Example 1 of the present invention 2- Confocal fluorescence imaging of HeLa cells before and after ionization. (A1, A2, A3) are cells incubated with 20.0 µM Cu-L for 1 hour, (B1, B2, B3) are cells incubated with 20.0 µM Cu-L for 1 hour, and then incubated with 100 µM S 2-ions for 1 hour, (C1, C2, C3) cells were incubated with 200 µM NEM for 1 hour, and then incubated with Cu-L (20 µM) for another 1 hour; A1, B1, C1 are fluorescence field transmission images, A2, B2, C2 are bright field transmission images, and A3, B3, C3 are merged transmission images (405-580 nm);

[0048] Fig.11 Comparison of the confocal fluorescence intensity before and after adding NEM and Cys to the probe Cu-L prepared in Example 1 of the present invention;

[0049] Fig.12 The confocal fluorescence imaging of HeLa cells before and after adding NEM and Cys to the probe Cu-L prepared in Example 1 of the present invention, wherein A1, A2, and A3 are cells incubated with 20.0 µM Cu-L for 1 hour, B1, B2, and B3 are cells incubated with 200 µM NEM for 1 hour, and then incubated with 20 µM Cu-L for another hour, C1, C2, and C3 are cells incubated with 200 µM NEM for 1 hour, and then incubated with 200 µM Cys for another hour, and finally, Cu-L (20 µM) is added and incubated for 1 hour; A1, B1, and C1 are fluorescence field transmission images, A2, B2, and C2 are bright field transmission images, and A3, B3, and C3 are merged transmission images (405-580 nm). DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0051] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0052] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0053] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0054] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0055] like Figure 1 As shown, the present invention provides a method for preparing a fluorescent probe for detecting H2S gas signal molecules, comprising the following steps:

[0056] S1: adding an organic solution of benzyl chloroformate dropwise to an organic solution of 1,4,7,10-tetraazacyclododecane, reacting at room temperature for 24-30 h, filtering the product, and recrystallizing it in ether to obtain the intermediate 1, wherein the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is (2-2.2):1;

[0057] S2: adding an organic solution of N,N-diisopropylethylamine (DIPEA) and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, reacting at 60-90° C. to obtain an intermediate 2, wherein the molar ratio of the intermediate 1 to triethylamine and tert-butyl bromoacetate is 1:(5-5.2):(3.3-3.5);

[0058] S3: Add a catalyst to the organic solution of the intermediate 2, and stir the reaction for 12 to 16 hours under a hydrogen atmosphere to obtain an intermediate 3. The catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and bromic acid / acetic acid (HBr / HOAc), and the mass fraction of palladium to carbon in the palladium-carbon catalyst is 5% to 10%, and the mass fraction of palladium hydroxide to carbon in the palladium hydroxide-carbon catalyst is 10% to 20%;

[0059] S4: Add potassium carbonate to the organic solution of the intermediate 3, and add an organic solution of dansyl chloride, and react for 12 to 16 hours to obtain the intermediate 4, wherein the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:(1.6 to 1.8):(2.2 to 2.4). Adding potassium carbonate in this step can neutralize the acid generated during the reaction and promote the reaction.

[0060] S5: adding trifluoroacetic acid to the organic solution of the intermediate 4, stirring the reaction for 12-16 h, and obtaining the fluorescent probe ligand L;

[0061] S6: Dissolve L in THF, adjust the pH value of the solution to 7-8 with sodium carbonate, add cupric chloride hexahydrate to the solution, and stir the reaction at 60-65°C for 4-5 hours to obtain the fluorescent probe Cu-L for detecting H2S gas signal molecules, wherein the molar ratio of the probe ligand L to cupric chloride hexahydrate is 1:(1.1-1.3).

[0062] The specific process is as follows:

[0063] S1: Synthesis of intermediate 1, i.e. 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane:

[0064] 1,4,7,10-tetraazacyclododecane was dissolved in chloroform, and benzyl chloroformate was dissolved in chloroform and slowly added dropwise to the chloroform solution of 1,4,7,10-tetraazacyclododecane under low temperature conditions, and then reacted at room temperature for 24-30 hours, and then filtered, the organic layer was evaporated and the solvent was concentrated, and it was recrystallized in ether to obtain intermediate 1. The slow dropwise addition method effectively controlled the reaction rate and avoided the formation of by-products;

[0065] S2: Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0066] 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane was dissolved in acetonitrile, and a solution of N,N-diisopropylethylamine (DIPEA) mixed in acetonitrile was added, and tert-butyl bromoacetate was dissolved in acetonitrile and added. The solution was heated at 60-90°C, the reaction was monitored by thin layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS), the solvent was concentrated, and the residue was extracted with ether and water. The organic layer was further washed with water, sodium hydroxide solution and water. The organic layer was dried over Mg2SO4 and the solvent was evaporated. The crude product was separated and purified by column chromatography to obtain intermediate 2;

[0067] S3: Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0068] Dissolve 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in anhydrous methanol, add the catalyst, and stir under H2 for 12-16 h. Remove the solvent and by-products under reduced pressure to obtain intermediate 3;

[0069] S4: Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0070] Dissolve 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in chloroform, add potassium carbonate, dissolve dansyl chloride (Cl-DNS) in chloroform, and stir at room temperature. Continue to react for 12-16 hours after the addition is complete. Cool to room temperature and filter. Wash the solution with saturated sodium bicarbonate solution and H2O in turn, collect the organic phase, add anhydrous sodium sulfate, leave overnight, filter, concentrate the solution, and purify the crude product with a silica gel column to obtain intermediate 4;

[0071] S5: Synthesis of probe ligand L, i.e., 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane:

[0072] Dissolve intermediate 4 in dichloromethane, add trifluoroacetic acid and mix at ambient temperature for 12-16 h. Add dichloromethane and remove the solvent, then add additional toluene and remove the solvent a second time to obtain the final probe ligand L;

[0073] S6: Synthesis of fluorescent probe Cu-L, i.e., Cu-1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane:

[0074] The probe ligand L was dissolved in tetrahydrofuran (THF), the pH of the solution was adjusted to 7-8 with sodium carbonate, copper chloride hexahydrate was added to the solution, and the reaction mixture was stirred at 60 °C for 4 h. The reaction mixture was then centrifuged and the organic phase was concentrated in vacuo. The fluorescent probe Cu-L was purified by flash chromatography (alumina, DCM / MeOH=30:1) to obtain a dark green solid.

[0075] The present invention designs the molecular structure of the fluorescent probe and utilizes the tert-butyl carbonyl on the derivative of Cyclen, 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane (DO2A), which is subsequently converted into a carboxyl group by acidification. The carboxyl group effectively enhances the chelating ability and water solubility of the compound, especially in the in vivo environment. The remaining secondary amine on DO2A is covalently coupled with DNS to obtain 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane, i.e., the probe ligand L. Due to Cu 2+ It has significant paramagnetism. The ligand L reacts with Cu 2+ The fluorescence is quenched when binding. When hydrogen sulfide is added to the Cu-L complex, S 2- With Cu 2+ The reaction forms a copper sulfide precipitate, which is released from the nitrogen azole macrocycle, resulting in fluorescence recovery. The present invention uses the Cyclen derivative containing the N-acetic acid side arm DO2A as a direct recognition group, which can coordinate copper ions. On this basis, the double-arm DNS is introduced as a connecting arm for other functional modifications. It has its unique advantages in constructing a fluorescent probe for detecting H2S gas signal molecules. Among them, the DNS group is a typical fluorophore with environment-dependent fluorescence emission, which promotes good potential candidates for the development of fluorescent probes. DNS is used as a linking arm to be substituted by nucleophilic substitution to the nitrogen heterocyclic DO2A ring. Based on the design and coordination between the above molecular parts, a fluorescent probe for detecting H2S gas signal molecules is designed. The fluorescent probe structure and experimental steps in the present invention enable the reaction to maintain a high efficiency and low by-product state in a dripping manner in the initial stage, and a high-purity product is successfully prepared. The probe is sensitive to the microenvironment, the system preparation is simple, the chemical stability is good, and the selectivity is good. It has high sensitivity, low detection limit and excellent selectivity for biological thiols, anions and amino acids. It also has low toxicity, high biosafety, and is suitable for intracellular H2S imaging. It can monitor intracellular H2S quickly, accurately, and specifically.

[0076] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0077] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0078] Example 1

[0079] A fluorescent probe for detecting H2S gas signal molecules and a preparation method and application thereof, comprising the following steps:

[0080] 1) Synthesis of intermediate 1, i.e. 1,7- N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane:

[0081] 1,4,7,10-Tetraazacyclododecane (1.0 g, 5.8 mmol) was dissolved in chloroform (30 mL) and cooled on ice. Benzyl chloroformate was dissolved in chloroform (10 mL) and slowly added dropwise to the above solution at 0°C for 7 h, and then the reaction mixture was stirred at room temperature (rt) for 24 h. The insoluble salts were filtered and the solvent was removed in vacuo to obtain a crude product. The crude product was washed with ether to obtain compound 1 as a white solid (2.1 g, yield 82%). 1 H NMR (600 MHz, DMSO-d) δ 7.33 (d, J = 4.7 Hz, 10H), 5.05 (s, 4H), 2.73 (s, 8H), 1.38 (s, 8H). MS (ESI, m / z) 441.33 [M+H] + This is consistent with the calculated molecular weight of 441.24.

[0082] 2) Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0083] Compound 1 (1.5 g, 3.4 mmol) was dissolved in ACN (20 mL), and then triethylamine (1.7 g, 17 mmol) was added. Subsequently, a solution of tert-butyl bromoacetate (2.17 g, 11.2 mmol) in acetonitrile (5 mL) was added and refluxed for 2 days. The reaction mixture was diluted with chloroform (25 mL), washed with water (20 mL), 1M hydrochloric acid (20 mL), 1M sodium hydroxide (20 mL) and water (20 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by column chromatography (alumina, DCM / MeOH = 50: 1-20: 1) to obtain a colorless oily compound (1.6 g, yield 70%). 1 H NMR (600 MHz, DMSO-d) δ 7.57 (s, 10H), 6.57 (d, J = 7.4 Hz, 4H), 4.24 (s, 4H), 2.80-2.73 (m, 8H), 2.58 (s, 8H), 2.04 (d, J = 19.3Hz, 18H). MS (ESI, m / z) 669.56.[M+H] + This is consistent with the calculated molecular weight of 669.38.

[0084] 3) Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0085] Compound 2 (1.5 g, 2.25 mmol) was dissolved in anhydrous methanol (10 mL), Pd-C (10% Pd, 0.55 g) was added, and stirred for 8 h in a hydrogen atmosphere. The solvent and by-products were removed by vacuum concentration. Compound 3 was obtained as a yellow oil (0.8 g, yield 89%). 1 H NMR (600 MHz, DMSO) δ 8.37 (s, 1H), 3.26 (s, 4H), 2.68 (s, 8H), 2.57-2.53 (m, 8H), 1.47 (s, 18H). MS (ESI, m / z) 401.33 [M+H] + This is consistent with the calculated molecular weight of 401.30.

[0086] 4) Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane:

[0087] Compound 3 (0.5 g, 1.25 mmol) was dissolved in CHCl3 (10 mL), K2CO3 (0.276 g, 2 mmol) and dansyl chloride (0.74 g, 2.75 mmol) were added, and the reaction mixture was stirred at rt overnight. At the end of the reaction, the insoluble salts were filtered and the solvent was removed in vacuo to obtain a crude oil residue. Purification by flash chromatography (Al2O3, DCM / MeOH=100:1-70:1) gave compound 4 (0.66 g, yield 61%) as a dark yellow powder. 1 H NMR (600 MHz, DMSO) δ8.54 (dt, J=8.6, 1.1 Hz, 2H), 8.30 (dt, J=8.7, 0.9 Hz, 2H), 8.10 (dd, J=7.4, 1.2 Hz, 2H), 7.69 (dd, J=8.5, 7.3 Hz, 2H), 7.64 (dd, J=8.7, 7.5 Hz, 2H), 7.30 (dd, J=7.7, 0.9 Hz, 2H), 3.45 (t, J=5.2Hz, 8H), 3.25 (s, 4H), 2H 2.88 (s, 12H), 2.56 (p, J=1.8 Hz, 8H), 1.40 (s, 18H). MS (ESI, m / z) 867.39[M+H] + This is consistent with the calculated molecular weight of 867.41.

[0088] 5) Synthesis of probe ligand L, i.e., 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane:

[0089] Compound 4 (0.5 g, 0.58 mmol) was dissolved in a mixture of DCM and TFA (3:1 v / v), and the reaction mixture was stirred at rt for 16 h. At the end of the reaction, 5 mL of methanol was added, and the mixture was concentrated in vacuo. Compound L was obtained as a dark yellow powder (0.39 g, yield 89%). 1 H NMR (600 MHz, DMSO-d) δ 8.54 (d, J=8.6 Hz, 2H), 8.30 (d, J=8.7 Hz, 2H), 8.06 (dd, J=7.4, 1.2 Hz, 2H), 7.68 (dd, J=8.5, 7.3 Hz, 2H), 7.63 (dd, J=8.7, 7.5 Hz, 2H), 7.28 (d, J=7.6 Hz, 2H), 3.87 (s, 4H), 3.55 (s, 8H), 3.32 (s, 7H), 2.83 (s, 12H)). 13C NMR (151 MHz, DMSO) δ170.41, 158.96, 151.43, 133.11, 130.71, 130.11, 129.76, 129.05, 124.34, 119.47, 116.17, 54.71, 46.30, 45.55. MS (ESI, m / z) 755.24[M+H] + This is consistent with the calculated molecular weight of 755.28.

[0090] 6) Synthesis of fluorescent probe Cu-L, i.e. Cu-1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane:

[0091] Ligand L (0.2 g, 0.27 mmol) was dissolved in tetrahydrofuran, and the pH of the solution was adjusted to 7-8 with Na2CO3. CuCl2·6H2O (0.05 g, 0.297 mmol) was added to the solution. The reaction mixture was stirred at 60°C for 4 h, and then the reaction mixture was centrifuged and the liquid organic phase was concentrated in vacuo. Purification by flash chromatography (Al2O3, DCM / MeOH=30:1) gave Cu-L as a dark green solid (0.187 g, yield 85%). 1H NMR (600 MHz, DMSO-d) δ8.98 (s, 4H), 8.07 (d, J=77.3 Hz, 6H), 7.51 (s, 2H), 3.38 (s, 20H), 3.00 (s, 12H).

[0092] In order to test and evaluate the performance of the fluorescent probe molecules obtained in the present invention, the following tests are used to illustrate.

[0093] First, a fluorescence titration experiment was conducted on the probe Cu-L prepared in Example 1 to investigate the effect of ligand L on the activity of different equivalents of Cu 2+ (0-1 eq) sensitivity, the test results are as follows Figure 2 As shown in the figure, as the Cu 2+ As the ion equivalent increases (0-1eq), the fluorescence intensity decreases. The detection limit is calculated by the following formula:

[0094]

[0095] The detection limit calculated from the titration results is 0.0441eq. Figure 2 It can be seen that the chelation of copper ions will affect the fluorescence properties of the ligand, and Figure 2 As shown in Figure A, L and Cu 2+ After binding, the fluorescence was quenched and accompanied by a slight red shift (47 nm), which may be attributed to the ligand field effect or steric hindrance effect.

[0096] Secondly, the H2S concentration response of the probe Cu-L prepared in Example 1 was tested to evaluate the quantitative detection capability and detection limit of the Cu-L probe.

[0097] The specific process is: add different concentrations (0~80 µM) of sodium sulfide to the 10 µM probe Cu-L solution (PBS buffer) for experiment, and then measure the fluorescence spectrum under 340 nm excitation. The test results are shown in Figure 3 As can be seen from the figure, no obvious emission band was observed in the fluorescence spectrum of the pure probe Cu-L solution. With the addition of sodium sulfide at different concentrations, an emission peak appeared at 562nm and gradually increased. The fluorescence intensity of the probe Cu-L in the range of 0~80 µM was positively correlated with the concentration of H2S ( Figure 3 The detection limit was calculated using the following formula:

[0098]

[0099] The calculated detection limit was 2.143 µM. The results showed that the Cu-L probe had high sensitivity to H2S and could be used to detect low-concentration H2S in biological systems.

[0100] Furthermore, the fluorescence intensity of the probe Cu-L prepared in Example 1 was tested over time. Specifically, the fluorescence spectrum of Cu-L was measured under 340 nm excitation, and the fluorescence intensity was measured over time (0-5 min) under the incubation conditions of sodium sulfide (60 µM) and 10 µM probe. The test results are shown in Figure 4 ,As can be seen from the figure, when Cu-L is added to H2S, the fluorescence is activated immediately (15s) and remains constant over time, indicating that the Cu-L probe has high sensitivity and continuous luminescence ,properties to H2S.

[0101] In order to verify the selectivity of the fluorescent probe prepared by the present invention, the selectivity of the probe Cu-L prepared in Example 1 to H2S was tested. In the selectivity experimental test, the test was carried out after incubation for one minute under the excitation condition of 340nm at room temperature. The selectivity of the Cu-L probe to various biothiols, sulfur-containing compounds, amino acids and anions (60 µM) and sodium sulfide (60 µM) was measured in PBS solution. Including threonine (Thr), glycine (Gly), tyrosine (Tyr), tryptophan (Trp), leucine (Leu), glutamic acid (Glu), lysine (Lys), cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), Ac - 、NO3 - , Cl - 、CO32- 、HCO3 - , HCO - 、SO4 2- 、SO3 2- 、HSO3 - 、S2O8 2- 、S2O3 2- and HS - The results are as follows Figures 5 to 7 As shown in the figure, when Cu-L is added to H2S, the fluorescence at 562 nm is significantly enhanced. Compared with other species, the probe Cu-L shows high selectivity for H2S. This further confirms that S 2- The CuS precipitate is formed by reaction with Cu, and the CuS precipitate is released from the nitrogen-containing macrocycle, resulting in the reactivation of fluorescence. As can be seen from the figure, other species do not cause significant changes in the fluorescence of the probe Cu-L. These results indicate that the Cu-L probe has good selectivity for H2S.

[0102] In order to verify the cytotoxicity of the fluorescent probe prepared in the present invention, the cytotoxicity and confocal imaging of the probe Cu-L prepared in Example 1 to H2S were tested. Specifically, the toxicity of Cu-L was evaluated using the MTT method. The test results are shown in FIG. Figure 8 As shown in the figure, when the concentration of Cu-L reached 50 μM, the viability of HeLa cells remained above 90-100%. Cytotoxicity tests showed that the Cu-L probe had low cytotoxicity and high biosafety.

[0103] Fig. 9 In order to add S to the probe Cu-L prepared in Example 1 of the present invention 2- Comparison of confocal fluorescence intensity before and after ionization shows that Cu-L is suitable for tracking the level of exogenous H2S in cells.

[0104] After confirming the sensitivity of Cu-L to H2S and its low cytotoxicity in vitro, the potential of probe Cu-L to locate H2S in cells was studied using HeLa cells as a model. Fig.10 , Fig.10 In order to add S to the probe Cu-L prepared in Example 1 of the present invention 2- Confocal fluorescence imaging of HeLa cells before and after ionization. (A1, A2, A3) are cells incubated with 20.0 µM Cu-L for 1 hour, (B1, B2, B3) are cells incubated with 20.0 µM Cu-L for 1 hour, and then incubated with 100 µM S 2-ions for 1 hour, (C1, C2, C3) cells were incubated with 200 µM NEM for 1 hour, and then incubated with Cu-L (20 µM) for another 1 hour; A1, B1, C1 are fluorescence field transmission images, A2, B2, C2 are bright field transmission images, A3, B3, C3 are merged transmission images (405~580nm). It can be seen from the figure that Cu-L (20 µM) cells show weak fluorescence in the green channel, which is due to the presence of a small amount of endogenous H2S. Compared with the control group, the S-containing cells 2- The ion probe has a significant fluorescence enhancement effect in the green channel ( Figure 8 ), which indicates that Cu-L is suitable for tracking the level of exogenous H2S in living cells.

[0105] Fig.11 This is a comparison of the confocal fluorescence intensity before and after adding NEM and Cys to the probe Cu-L prepared in Example 1 of the present invention. It can be seen from the figure that Cu-L has good biological imaging capabilities for both exogenous cells and endogenous cells.

[0106] Fig.12 This is the confocal fluorescence imaging of HeLa cells before and after adding NEM and Cys to the probe Cu-L prepared in Example 1 of the present invention, wherein A1, A2, and A3 are cells incubated with 20.0 µM Cu-L for 1 hour, B1, B2, and B3 are cells incubated with 200 µM NEM for 1 hour, and then incubated with 20 µM Cu-L for another hour, C1, C2, and C3 are cells incubated with 200 µM NEM for 1 hour, and then incubated with 200 µM Cys for another hour, and finally, Cu-L (20 µM) was added and incubated for 1 hour; A1, B1, and C1 are fluorescence field transmission images, A2, B2, and C2 are bright field transmission images, and A3, B3, and C3 are merged transmission images (405-580 nm). Fig.11 The endogenous imaging results showed that the probe Cu-L exhibited very weak fluorescence in cells. The fluorescence of the inhibitor group treated with NEM was reduced to a negligible level, indicating that endogenous H2S that may exist in the cells has been successfully eliminated. After the addition of inhibitors NEM and Cys, the fluorescence intensity increased, indicating that Cu-L has good bioimaging capabilities for both exogenous and endogenous cells.

[0107] Example 2

[0108] A method for preparing a fluorescent probe for detecting H2S gas signal molecules comprises the following steps:

[0109] S1: adding an organic solution of benzyl chloroformate dropwise to an organic solution of 1,4,7,10-tetraazacyclododecane, reacting at room temperature for 24 h, filtering the product, and recrystallizing in ether to obtain the intermediate 1, wherein the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is 2:1;

[0110] S2: adding an organic solution of DIPEA and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, reacting at 60° C. to obtain an intermediate 2, wherein the molar ratio of the intermediate 1 to triethylamine and tert-butyl bromoacetate is 1:5:3.3;

[0111] S3: Add palladium hydroxide-carbon catalyst to the organic solution of the intermediate 2, and stir the reaction for 12 to 16 hours under a hydrogen atmosphere to obtain an intermediate 3. In the palladium hydroxide-carbon catalyst, the mass fraction of palladium hydroxide to carbon is 10%;

[0112] S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and reacting for 12 h to obtain the intermediate 4, wherein the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:1.6:2.2.

[0113] S5: adding trifluoroacetic acid to the organic solution of the intermediate 4, stirring and reacting for 12 h to obtain the fluorescent probe ligand L;

[0114] S6: Dissolve L in THF, adjust the pH value of the solution to 7 with sodium carbonate, add cupric chloride hexahydrate to the solution, and stir the reaction at 60°C for 5 h to obtain the fluorescent probe Cu-L for detecting H2S gas signal molecules, wherein the molar ratio of the probe ligand L to cupric chloride hexahydrate is 1:1.1.

[0115] Example 3

[0116] A method for preparing a fluorescent probe for detecting H2S gas signal molecules comprises the following steps:

[0117] S1: adding an organic solution of benzyl chloroformate dropwise to an organic solution of 1,4,7,10-tetraazacyclododecane, reacting at room temperature for 30 h, filtering the product, and recrystallizing in ether to obtain the intermediate 1, wherein the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is 2.2:1;

[0118] S2: adding an organic solution of DIPEA and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, reacting at 90° C. to obtain an intermediate 2, wherein the molar ratio of the intermediate 1 to triethylamine and tert-butyl bromoacetate is 1:5.2:3.5;

[0119] S3: Adding palladium-polyethyleneimine catalyst to the organic solution of the intermediate 2, stirring and reacting for 12 to 16 hours under a hydrogen atmosphere to obtain the intermediate 3.

[0120] S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and reacting for 16 hours to obtain the intermediate 4, wherein the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:1.8:2.4.

[0121] S5: adding trifluoroacetic acid to the organic solution of the intermediate 4, stirring and reacting for 16 h to obtain the fluorescent probe ligand L;

[0122] S6: Dissolve L in THF, adjust the pH value of the solution to 8 with sodium carbonate, add cupric chloride hexahydrate to the solution, and stir the reaction at 65°C for 4 hours to obtain the fluorescent probe Cu-L for detecting H2S gas signal molecules, wherein the molar ratio of the probe ligand L to cupric chloride hexahydrate is 1:1.3.

[0123] Example 4

[0124] A method for preparing a fluorescent probe for detecting H2S gas signal molecules comprises the following steps:

[0125] S1: adding an organic solution of benzyl chloroformate dropwise to an organic solution of 1,4,7,10-tetraazacyclododecane, reacting at room temperature for 28 h, filtering the product, and recrystallizing in ether to obtain the intermediate 1, wherein the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is 2:1;

[0126] S2: adding an organic solution of DIPEA and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, reacting at 80° C. to obtain an intermediate 2, wherein the molar ratio of the intermediate 1 to triethylamine and tert-butyl bromoacetate is 1:5:3.4;

[0127] S3: adding palladium-polyethyleneimine / formic acid catalyst to the organic solution of the intermediate 2, stirring and reacting for 12 to 16 hours under a hydrogen atmosphere to obtain the intermediate 3;

[0128] S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and reacting for 15 hours to obtain the intermediate 4, wherein the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:1.7:2.3.

[0129] S5: adding trifluoroacetic acid to the organic solution of the intermediate 4, stirring and reacting for 15 h to obtain the fluorescent probe ligand L;

[0130] S6: Dissolve L in THF, adjust the pH value of the solution to 8 with sodium carbonate, add cupric chloride hexahydrate to the solution, and stir the reaction at 62°C for 4.5 h to obtain the fluorescent probe Cu-L for detecting H2S gas signal molecules, wherein the molar ratio of the probe ligand L to cupric chloride hexahydrate is 1:1.2.

[0131] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A copper ion-containing bioluminescent probe for detecting endogenous H2S in cells, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells as claimed in claim 1, characterized in that: The following steps are involved: S1: adding a chloroform solution of benzyl chloroformate dropwise to a chloroform solution of 1,4,7,10-tetraazacyclododecane, and reacting at room temperature to obtain intermediate 1; The structural formula of the intermediate 1 is: ; S2: adding triethylamine and acetonitrile solution of tert-butyl bromoacetate to the acetonitrile solution of the intermediate 1 in sequence, and refluxing and stirring to react to obtain the intermediate 2; The structural formula of the intermediate 2 is: ; S3: adding a catalyst to the methanol solution of the intermediate 2, stirring and reacting under a hydrogen atmosphere to obtain an intermediate 3; The structural formula of the intermediate 3 is: ; S4: adding potassium carbonate to the chloroform solution of the intermediate 3, and adding an organic solution of dansyl chloride, and after the reaction is completed, obtaining the intermediate 4; The structural formula of the intermediate 4 is: ; S5: adding the intermediate 4 to a mixed solution of dichloromethane and trifluoroacetic acid, stirring for reaction, and obtaining a fluorescent probe ligand L; The structural formula of the fluorescent probe ligand L is: ; S6: dissolving the fluorescent probe ligand L in THF, adjusting the pH value of the solution to 7-8, then adding copper chloride, stirring the reaction, and preparing the copper ion-containing biological fluorescent probe for detecting endogenous H2S in cells; The reaction temperature in step S2 is 60-90°C and the reaction time is 42-48 h; In step S4, the molar ratio of the intermediate 3 to potassium carbonate and dansyl chloride is 1:(1.6-1.8):(2.2-2.4).

3. The method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells according to claim 2, characterized in that: In step S1, the molar ratio of benzyl chloroformate to 1,4,7,10-tetraazacyclododecane is (2-2.2):

1.

4. The method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells according to claim 2, characterized in that: In step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and bromic acid / acetic acid. For the palladium-carbon catalyst, the mass fraction of palladium to carbon is 5% to 10%, and for the palladium hydroxide-carbon catalyst, the mass fraction of palladium hydroxide to carbon is 10% to 20%.

5. The method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells according to claim 2, characterized in that: In step S5, the reaction time is 12 to 16 h.

6. The method for preparing a copper ion-containing bioluminescent probe for detecting endogenous H2S in cells according to claim 2, characterized in that: In step S6, the reaction temperature is 60-65° C., the reaction time is 4-5 h; and the molar ratio of the probe ligand L to copper chloride is 1:(1.1-1.3).

7. Use of the copper ion-containing bioluminescent probe described in claim 1 in preparing a kit for detecting endogenous H2S in cells.

8. A kit for detecting endogenous H2S in cells, characterized in that: The invention comprises the copper ion-containing bioluminescent probe for detecting endogenous H2S in cells as described in claim 1.

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