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| Targets |
The target is nitroreductase (NTR), an enzyme that is overexpressed in hypoxic tumor cells and in the hypoxic kidney. Nitroreductase is an enzyme that reduces nitro groups to amino groups. The probe's structure contains a nitro group that acts as a quencher. Upon enzymatic reduction by NTR, the probe produces a strong fluorescent signal.
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| ln Vitro |
In living HeLa cells with reduced oxygen concentration, 3-MeOARh-NTR (10 μM, 30 min; cells treated in 20% O2 and 10% O2 for 12 h) produces a robust fluorescence signal [1]. 3. Fluorescence imaging of mouse kidney tissue (λex=488 nm; λem=510-590 nm) was created by MeOARh-NTR (10 μM, 30 min). 3-MeOARh-NTR is a useful probe for measuring NTR-based renal hypoxia [1].
In vitro, 3-MeOARh-NTR acts as a highly selective "off-on" fluorescent probe. In the absence of NTR, its fluorescence is quenched. Upon incubation with NTR, the nitro group is reduced, leading to a strong increase in fluorescence. At a concentration of 10 microM with a 30-minute incubation time, it produces a strong fluorescent signal in live HeLa cells. |
| ln Vivo |
In vivo, 3-MeOARh-NTR is an effective probe for detecting and evaluating renal hypoxia via NTR assessment. When administered systemically (e.g., intravenously), the probe accumulates in the kidneys. In a state of renal hypoxia (such as in acute kidney injury), NTR activity increases, leading to a proportional increase in fluorescence intensity in the tissue. This allows real-time imaging of hypoxia.
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| Enzyme Assay |
A non-cell enzymatic assay is performed to confirm NTR activity. Purified recombinant NTR is incubated with the probe (10 uM) in assay buffer (PBS, pH 7.4) in the presence of NADH (a cofactor). Fluorescence intensity is measured over time using a microplate reader (ex/em 490/525 nm). An increase in fluorescence indicates probe reduction by NTR.
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| Cell Assay |
For cellular imaging, HeLa or other cancer cells are seeded in confocal dishes. Cells are treated with 3-MeOARh-NTR (10 microM) for 30 minutes. To induce NTR expression, cells may be pre-treated with hypoxia-mimetic agents (e.g., CoCl2). After washing, live-cell fluorescence imaging is performed. A strong green fluorescence signal is observed in hypoxic cells, indicating successful activation of the probe.
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| Animal Protocol |
In vivo imaging is performed in a mouse model of renal ischemia-reperfusion injury (IRI). Mice are administered 3-MeOARh-NTR (1-5 mg/kg) via intravenous (IV) or intraperitoneal (IP) injection. After 1-2 hours, the mice are anesthetized, and the kidneys are imaged using an in vivo imaging system (IVIS) with appropriate excitation/emission filters. Fluorescence intensity correlates with the severity of renal hypoxia.
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| ADME/Pharmacokinetics |
The compound has a molecular weight of 596.61. It is soluble in DMSO at 5 mg/mL (with ultrasonic and warming). For in vivo imaging, the probe is typically formulated in a mixture of DMSO and PBS or saline. It is stable as a solid at -20degC for up to 3 years. The probe produces stable fluorescence after activation, allowing for reliable imaging.
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| Toxicity/Toxicokinetics |
As a research reagent, toxicity data is limited. The compound is generally non-toxic at the imaging concentrations (10 microM in vitro, 1-5 mg/kg in vivo). The probe is not intended for human therapeutic use. Standard safety precautions for handling fluorescent dyes should be followed. No genotoxicity has been reported for this class of probes.
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| References | |
| Additional Infomation |
3-MeOARh-NTR is a research-grade chemical tool, not a drug or clinical therapeutic. It is used as an activatable imaging probe for detecting and evaluating renal hypoxia. The compound has no FDA approval. It is for research use only (RUO) and is a valuable molecular tool for studying hypoxia in cancer and kidney disease.
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| Molecular Formula |
C33H30N3O8+
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| Molecular Weight |
596.61
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| Related CAS # |
3-MeOARh-NTR chloride
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| Appearance |
Pink to orange solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :~5 mg/mL (~8.38 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6761 mL | 8.3807 mL | 16.7614 mL | |
| 5 mM | 0.3352 mL | 1.6761 mL | 3.3523 mL | |
| 10 mM | 0.1676 mL | 0.8381 mL | 1.6761 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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