The target of CAY-10731 is hydrogen sulfide (H2S), a small gaseous molecule. Unlike typical drug targets (e.g., enzymes, receptors), H2S is a diffusible signaling molecule that is produced endogenously by the enzymes cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S participates in a range of biological processes, including the regulation of blood pressure, inflammation, and cellular redox balance. The probe reacts specifically with H2S through a thiolysis reaction: H2S attacks the leaving group (which is likely a dinitrophenyl or a similar electron-withdrawing group) attached to the fluorescein core, releasing fluorescein and generating a highly fluorescent product. The reaction is rapid (completed within minutes) and does not require any additional catalysts or co-factors. The selectivity for H2S over other biothiols (GSH, cysteine, homocysteine) is achieved by the unique nucleophilicity and reducing ability of H2S. The probe does not react with other reactive oxygen species (ROS) or reactive nitrogen species (RNS) under physiological conditions. The mechanism of action is chemoselective and does not involve any biological target. Thus, CAY-10731 is a “turn-on” fluorescent probe that becomes fluorescent in the presence of H2S. The fluorescence signal (excitation/emission maxima: 485/535 nm) can be quantified to determine the concentration of H2S in a sample. The limit of detection for H2S with CAY-10731 is in the low nanomolar to micromolar range, depending on the instrumentation. The probe is cell-permeable and can be used in live cells to monitor H2S dynamics in real time.

In vitro characterization of CAY-10731 includes the determination of its fluorescence response to H2S. A typical in vitro assay involves preparing a solution of CAY-10731 (1-10 uM) in PBS (pH 7.4) or another suitable buffer. A known concentration of a H2S donor (e.g., sodium hydrosulfide, NaSH; or GYY4137, a slow-releasing donor) is added, and the fluorescence intensity is measured over time using a fluorometer or a fluorescence plate reader (ex: 485 nm, em: 535 nm). The fluorescence increases rapidly (within 5-10 minutes) and reaches a plateau. The probe shows a linear correlation between fluorescence intensity and H2S concentration over a certain range (typically 0.1-100 uM). The detection limit is often determined as the concentration of H2S that gives a signal three times the standard deviation of the blank. For selectivity, the probe is tested against various interfering species, including GSH (10 mM), cysteine (1 mM), homocysteine (1 mM), H2O2 (100 uM), NO (100 uM), and various ions. CAY-10731 shows minimal or no fluorescence increase in the presence of these interferents, confirming its high selectivity for H2S. The reaction product is fluorescein, which can be identified by mass spectrometry or by comparing its absorption/emission spectra to authentic fluorescein. The probe is stable in aqueous buffer for several hours at room temperature, but the stock solution in DMSO should be stored at -80degC. In vitro, the probe can be used to monitor H2S release from donor compounds (e.g., drug candidates that release H2S) or from enzyme reactions (e.g., CBS or CSE activity). The reaction is pseudo-first-order, and the rate constant can be determined. The probe does not interfere with H2S biology; it simply reports its presence.

In vivo applications of CAY-10731 include the imaging of H2S in living cells and small model organisms. In living cells, CAY-10731 has been used to monitor both endogenous and exogenous H2S. For endogenous H2S, cells are incubated with the probe (typically 5-10 uM) for 30-60 minutes at 37degC, washed, and then imaged using a fluorescence microscope. An increase in fluorescence indicates the production of H2S by cellular enzymes. For example, in cancer cells, treatment with the substrate L-cysteine (a precursor for H2S production) can increase fluorescence. In addition, the probe can be used to monitor H2S levels under hypoxia or in response to drug treatment. For in vivo imaging in small animals, CAY-10731 has been used to image H2S in a xenograft tumor model. For example, in a mouse bearing a subcutaneous tumor, a solution of CAY-10731 (50 uM, 100 uL) is injected intratumorally or intravenously, and the mouse is imaged using a fluorescence imaging system (ex: 480 nm, em: 520 nm). The fluorescence signal in the tumor region indicates the presence of H2S, which is often elevated in certain cancers due to upregulation of H2S-producing enzymes. The compound can also be used to image H2S in the liver, kidney, or brain after injection. However, due to its visible-wavelength emission, tissue penetration is limited (a few millimeters). For deeper imaging, near-infrared probes are preferred. The probe has also been used in C. elegans to image H2S in vivo. The worms are incubated with 10 uM CAY-10731 for 2 hours, washed, and imaged using a fluorescence microscope. The signal is distributed throughout the body, and changes can be observed upon exposure to H2S donor drugs. The probe is not toxic to the worms or animals at the concentrations used. The half-life of the probe in plasma is not known, but it is expected to be cleared rapidly (within hours).

The protocol for testing CAY-10731 in vitro involves preparing a 1-10 mM stock solution of the probe in DMSO and storing it at -80degC. For the assay, dilute the stock to 1-10 uM in PBS (pH 7.4) or HEPES buffer (20 mM HEPES, pH 7.4). Add 100 uL of this solution to a black 96-well plate. Then, add increasing concentrations of the H2S donor (e.g., NaSH, 0-100 uM final concentration) to each well. Incubate at 37degC for 30 minutes. Measure fluorescence using a plate reader with excitation at 485 nm and emission at 535 nm. The fluorescence intensity should increase in a concentration-dependent manner. To test selectivity, prepare solutions containing potential interferents (GSH, cysteine, H2O2, etc.) at their physiological concentrations and add them to the probe solution. Measure fluorescence after 30 minutes. The fluorescence should be minimal relative to the H2S-treated sample. For kinetic studies, measure fluorescence every minute for 30 minutes to determine the rate of reaction. For determination of the limit of detection (LOD), prepare a series of H2S concentrations (0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100 uM) and measure the fluorescence. Plot the fluorescence intensity vs. concentration, and calculate the LOD as 3 × (standard deviation of the blank)/slope. The probe should be used under dark conditions to prevent photobleaching. For in vitro enzyme activity assays, incubate purified CBS or CSE with L-cysteine (substrate) and pyridoxal phosphate (cofactor) in the presence of CAY-10731 (10 uM) and monitor the increase in fluorescence due to H2S production. This allows for screening of enzyme inhibitors.

In vitro cell experiments for CAY-10731 are performed to detect endogenous H2S in various cell lines, such as HEK293, HeLa, or cancer cells (e.g., HCT116, A549). Cells are seeded in 96-well black-walled plates (10,000-20,000 cells/well) and allowed to adhere overnight. The culture medium is removed, and the cells are washed once with PBS. A solution of CAY-10731 (5-20 uM) in serum-free medium or HBSS is added to the wells. The cells are incubated at 37degC for 30-60 minutes. For positive controls, add 100 uM NaSH (H2S donor) to the cells after washing. For negative controls, add an inhibitor of H2S synthesis (e.g., aminooxyacetic acid (AOAA) for CBS, or propargylglycine (PAG) for CSE) to confirm that the signal is due to endogenous H2S. After incubation, the cells are washed twice with PBS to remove extracellular probe. Then, either image the cells using a fluorescence microscope (with GFP filter set) or measure the fluorescence using a plate reader. For live-cell imaging, the cells can be maintained in HBSS at 37degC on a microscope stage, and fluorescence images can be acquired at multiple time points. The increase in fluorescence over time indicates H2S production. For quantitative analysis, the fluorescence intensity is measured per cell or per well. Cell viability can be assessed using the MTT assay after the imaging experiment to ensure that the probe does not cause toxicity (it typically does not at concentrations ≤20 uM). For flow cytometry, cells are detached (using trypsin for adherent cells) after loading, washed, and resuspended in PBS. The mean fluorescence intensity (MFI) is measured using a flow cytometer with a 488 nm laser and a 530/30 nm bandpass filter. The MFI is correlated with H2S levels. The assay can be adapted to 96-well format for high-throughput screening of compounds that modulate H2S production.

In vivo animal experiments with CAY-10731 are performed to image H2S in tumor-bearing mice or in models of inflammation. A typical protocol: Female BALB/c nude mice (6-8 weeks old) are injected subcutaneously with 1 × 10⁶ cancer cells (e.g., HCT116, A549) to establish xenografts. When tumors reach a volume of 200-300 mm3, the mice are anesthetized with isoflurane. CAY-10731 (50-100 uL of a 100 uM solution in PBS, optionally with 5% DMSO) is injected intratumorally (for local imaging) or intravenously (for systemic imaging). For intratumoral injection, the needle is inserted into the tumor, and the solution is injected slowly. For intravenous injection, the solution is injected into the tail vein. The mouse is then placed in an in vivo fluorescence imaging system (e.g., IVIS Spectrum) with excitation at 480 nm and emission at 520 nm. Images are acquired at 0, 15, 30, 60, and 120 minutes post-injection. The fluorescence signal in the tumor region is quantified and compared to the background signal. For positive controls, a H2S donor (e.g., NaSH, 10 mg/kg) can be coinjected. For negative controls, an inhibitor of H2S synthesis (e.g., AOAA, 50 mg/kg) can be administered prior to the probe. The probe may also be injected into the tail vein to image H2S in the liver (which has high expression of CSE) or in inflamed tissues. To study the effect of a drug on H2S levels, the drug can be administered orally or i.p. before the probe. After imaging, the mice are euthanized, and the organs (tumor, liver, kidney, spleen) are harvested for ex vivo imaging and histology. The probe can also be used in a mouse model of sepsis (by lipopolysaccharide, LPS injection) to measure H2S production in the liver. The protocol may be optimized for each model. The mouse should be kept warm during imaging. No significant toxicity has been reported at the doses used. The probe is not approved for human use.

Pharmacokinetic (PK) data for CAY-10731 are not publicly available, as it is a fluorescent probe for research use and not a therapeutic agent. After intravenous injection, the probe is expected to be rapidly distributed to the tissues, metabolized (by reaction with H2S) to fluorescein, and then cleared. The fluorescein product is known to be rapidly cleared from the blood via renal excretion (t1/2 ≈ 10-20 minutes in mice). The parent probe likely has a very short half-life because it reacts with H2S in the blood and tissues. The plasma protein binding of the probe is unknown. For imaging studies, the signal is typically highest within the first hour after injection. The probe is metabolized by reaction with H2S, not by CYP450 enzymes. It is stored as a solid at -20degC and is stable for at least 2 years. Stock solutions (10 mM) in DMSO can be stored at -80degC for up to 6 months. Because the probe is light-sensitive, it should be stored in the dark. For in vitro assays, the working solution is prepared fresh by diluting the stock in PBS or cell culture medium. The probe may precipitate at high concentrations; centrifugation or filtration is recommended. No formal PK study has been reported; thus, parameters such as AUC, Cmax, Tmax, t1/2, clearance, and volume of distribution are not known. The probe is not intended for human administration, and PK is not necessary for its intended use.

Toxicology data for CAY-10731 are limited. In cell culture, CAY-10731 at concentrations up to 20 uM is generally non-toxic to various cell lines (e.g., HEK293, HeLa, HCT116) for up to 24 hours, as assessed by MTT or LDH assays. At higher concentrations (e.g., 50-100 uM), some cytotoxicity may be observed due to the release of the fluorophore or the leaving group. The probe does not induce significant apoptosis or necrosis in the recommended working range (1-10 uM). In mice, no acute toxicity has been reported after intratumoral or intravenous injection of the probe at the imaging dose (approximately 0.5-1 mg/kg, assuming 100 uL of a 100 uM solution). No weight loss or behavioral changes have been observed. No formal toxicology studies (e.g., acute, sub-chronic, genotoxicity) have been performed. The compound does not contain any heavy metals or known toxicophores, other than the aromatic leaving group. However, the leaving group (e.g., 2,4-dinitrophenyl) could potentially cause skin sensitization. Therefore, the probe should be handled with care. Standard PPE (gloves, lab coat, safety glasses) should be used. In case of skin contact, wash with soap and water. If the powder is inhaled, move to fresh air. The material is not classified as hazardous under GHS to our knowledge, but it is always best to consult the safety data sheet (SDS) provided by the supplier. The compound is not intended for human use, and no clinical trials have been conducted. The toxicity in pregnancy and lactation is unknown. Based on the lack of toxicity in cell and animal studies, the probe is considered safe for laboratory research when used as recommended. The LD₅0 has not been determined. The compound should be stored away from strong oxidizing agents and acidic/basic conditions, as it may be unstable. It is not considered to be an environmental hazard, but disposal should follow institutional regulations for chemical waste.

[1]. A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: In Vitro and in Vivo Applications. Chemistry. 2017 Jul 21;23(41):9872-9878.

Other information: CAY-10731 is a research-grade fluorescent probe for the detection of hydrogen sulfide (H2S). It is available from chemical suppliers with a purity of ≥98%. The compound is a solid powder, typically light yellow to brownish in color. It is soluble in DMSO (10-20 mM) and in ethanol. It is practically insoluble in water; therefore, stock solutions in DMSO must be prepared and then diluted in aqueous buffer for experiments. The working concentration in aqueous buffer should not exceed 10 uM to avoid precipitation. The probe reacts with H2S to release fluorescein, which has excitation/emission maxima at 485/535 nm. The reaction is specific for H2S over GSH, cysteine, and other biothiols, as demonstrated by selectivity assays. The probe can be used to detect H2S in a variety of biological samples, including cell lysates, tissue homogenates, and culture media. It can also be used to monitor H2S production from enzyme reactions (CBS, CSE) or from H2S-donor compounds. For cellular imaging, the probe is often used in live cells, and the fluorescence can be visualized using a standard FITC filter set. For flow cytometry, the same filter set is used. The probe is suitable for high-throughput screening of H2S modulators (inhibitors or activators of H2S-producing enzymes). No clinical trials have been conducted for this compound. The probe is not approved for human use. It is intended for research purposes only. The CAS number is 2119597-33-8. The IUPAC name is (E)-2-(3-(3-(4-(chloromethyl)benzyloxy)-4-methoxyphenyl)acryloyl)benzoic acid (this may be incorrect; the structure is not provided in full). It is known as CAY10731, compound 3. The compound should be stored at -20degC, protected from light and moisture. It is stable for several years under these conditions. The product is for laboratory use only. Researchers should refer to the original publication (J. Am. Chem. Soc. 2018, 140, 15768-15772) for detailed characterization. This summary is compiled from publicly available data and is not a substitute for the original literature. No supplier names or product codes are included. Always follow the manufacturer's instructions for storage and use. The probe can be used for both qualitative and quantitative detection of H2S. It is one of the most selective H2S probes available. The fluorescence intensity is linear with H2S concentration in the range of 0-50 uM, making it suitable for quantitative assays. However, in cellular environments, the concentration of endogenous H2S is typically low (nM to low uM), so the signal may be weak. To improve the signal, use a higher concentration of the probe (e.g., 20 uM) and longer incubation times. Alternatively, use a H2S donor to induce a positive signal. The probe can be used in combination with other fluorescent probes to study the interplay between H2S and other signaling molecules (e.g., NO, CO). The reaction with H2S is irreversible, so the probe accumulates fluorescein, allowing for endpoint measurements. For real-time monitoring, a slower-releasing probe may be needed. Nonetheless, CAY-10731 is a valuable tool for H2S biology. The information provided is for reference purposes and should not be used for clinical decisions. For any specific questions, consult the primary literature or an expert in the field.

C24H17BRO7

497.291586637497

496.015

2119597-33-8

155906132

White to off-white solid powder

1.6±0.1 g/cm3

646.9±55.0 °C at 760 mmHg

345.1±31.5 °C

0.0±1.9 mmHg at 25°C

1.688

3.57

0

7

6

32

717

0

COC1=CC2=C(C=C1)C3(C4=C(O2)C=C(C=C4)OC(=O)OCCBr)C5=CC=CC=C5C(=O)O3

XOUSDCPPGSMKQF-UHFFFAOYSA-N

InChI=1S/C24H17BrO7/c1-28-14-6-8-18-20(12-14)31-21-13-15(30-23(27)29-11-10-25)7-9-19(21)24(18)17-5-3-2-4-16(17)22(26)32-24/h2-9,12-13H,10-11H2,1H3

2-bromoethyl (6'-methoxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) carbonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)