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| Targets |
TRFS-green targets the selenoprotein thioredoxin reductase (TrxR), an important enzyme in cellular redox regulation. The probe itself is a substrate for TrxR, which reduces its disulfide bond. This reduction triggers a specific chemical reaction (intramolecular cyclization) that results in the unmasking of a naphthalimide fluorophore, leading to a strong fluorescent signal. This makes it a highly specific tool for detecting TrxR activity.
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| ln Vitro |
TrxR-mediated disulfide bond breaking and subsequent intramolecular cyclization liberate the veiled naphthalimide fluorophore, causing TRFS-green to exhibit a green fluorescence switching change [1]. TRFS-green exhibits a faint signal at about 480 nm when stimulated at 377 nm. The creation of live cell-based TrxR inhibitor screening assays is aided by the pretreatment of living cells with TrxR inhibitors in a dose-dependent manner, which can block the fluorescence signal of TRFS-green in living cells [1]. The two main thiols in cells, GSH and Cys, as well as endogenous reducing agents like vitamin C and NADPH, have no effect on TRFS-green [1]. Assess TrxR activity in crude cell extracts [1]: Use a fluorometer (Em=540 nm, Ex=440 nm) to assess the fluorescence intensity after incubating cell lysates with TRFS-green for the recommended amount of time (about 1-2 hours). Moreover, the time-dependent increase in fluorescence over a certain duration (Em=540 nm, Ex=440 nm) can be observed. Through the microscope's green fluorescence channel, cells treated with 10 μM with TRFS-green for 2-4 hours can be seen [1]. Note: It is advised to wash the cells with PBS or new medium right before taking pictures in order to remove any remaining TRFS-green in order to achieve clean fluorescence images. The first fluorescent probe for TrxR with green emission is called TRFS-green. Nevertheless, TRFS-green reacts slowly to enzymes, and it takes more than two hours to produce the maximum fluorescence signal—even in the case of reducing agent tris(2-carboxyethyl)phosphine (TCEP) [2].
In vitro, TRFS-green demonstrates high selectivity for TrxR over other related enzymes and over abundant cellular reducing agents like glutathione (GSH), cysteine (Cys), NADPH, and vitamin C. The probe has a maximum absorbance at around 373 nm and exhibits a green fluorescence upon activation, with an emission signal around 480 nm. It has a moderate fluorescence fold increase (∼30-fold) and a relatively slow response time, often requiring 2 hours or more to reach maximal signal. |
| ln Vivo |
In vivo, the activity of TRFS-green is observed in real-time as it images TrxR activity in living cells. It can be used in live-cell imaging to visualize where and how strongly TrxR is active. For example, if cells are pre-treated with a TrxR inhibitor, the fluorescence signal from TRFS-green is decreased in a dose-dependent manner, validating its specificity. This allows researchers to monitor TrxR activity in response to various drugs or genetic manipulations in a cellular context.
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| Enzyme Assay |
TRFS-green is used in cell-free biochemical assays to assess TrxR activity in lysates or protein extracts. The probe is incubated with the sample, and the increase in fluorescence (excitation ~377 nm, emission ~480 nm) is measured over time using a fluorescence microplate reader or spectrophotometer. The rate of fluorescence increase is directly proportional to the TrxR activity in the sample, allowing for the quantification of enzyme activity or the screening of potential TrxR inhibitors in a high-throughput format.
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| Cell Assay |
For cell-based experiments, cells are seeded in culture dishes or multi-well plates suitable for fluorescence imaging. TRFS-green is added to the cell culture medium (typically at a concentration of 1-10 uM) and incubated for a period of time (e.g., 2 hours or more). After incubation, the cells are washed with PBS to remove excess probe, and then imaged using a fluorescence microscope or a plate reader with appropriate filter sets (e.g., excitation ~377 nm, emission ~480 nm). The green fluorescence intensity observed correlates with the level of active TrxR in the cells.
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| Animal Protocol |
TRFS-green is not typically used in whole-animal studies. However, it can be used in ex vivo assays on tissue samples collected from animals. For example, from an animal model of a disease, tissues such as the brain, liver, or tumor can be harvested, homogenized, and the lysate can be used in a cell-free activity assay as described above. This allows researchers to measure TrxR enzyme activity in the tissue, which can be correlated with the disease state or treatment effect.
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| ADME/Pharmacokinetics |
TRFS-green is a chemical probe and not a drug, so it does not have classical pharmacokinetic parameters. In cell culture, its uptake and activation are time-dependent, with maximal signal often achieved after 2 hours or more. The probe is cell-permeable and stable in the culture medium. It should be stored as a powder at -20degC, protected from light, and dissolved in an organic solvent like DMSO to prepare a stock solution, which is then diluted in an aqueous buffer for experiments.
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| Toxicity/Toxicokinetics |
TRFS-green is a research chemical and is not intended for therapeutic use. Specific toxicological data is not extensively documented. However, it is designed to be a low-toxicity fluorescent probe for live-cell imaging. Standard laboratory safety precautions for handling fluorescent dyes and chemical reagents, including the use of gloves, lab coats, and safety glasses, should be observed.
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| References | |
| Additional Infomation |
TRFS-green is not a drug but a highly specialized fluorescent probe for research. It is used to study the biology of thioredoxin reductase, a key enzyme in the cellular antioxidant defense system. It is a powerful tool for understanding the role of TrxR in cancer, neurodegenerative diseases, and other conditions involving oxidative stress. Its selectivity for TrxR makes it a superior alternative to less specific redox probes for studying this particular enzyme.
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| Molecular Formula |
C20H20N2O4S2
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| Molecular Weight |
416.513802528381
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| Exact Mass |
416.086
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| CAS # |
1513848-14-0
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| PubChem CID |
73670302
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| Appearance |
White to off-white solid powder
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| LogP |
3.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
625
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1CC(CS1)OC(NC1=CC=C2C(N(C(C3C=CC=C1C2=3)=O)CCCC)=O)=O
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| InChi Key |
MUAYCZWBPDUZKR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H20N2O4S2/c1-2-3-9-22-18(23)14-6-4-5-13-16(8-7-15(17(13)14)19(22)24)21-20(25)26-12-10-27-28-11-12/h4-8,12H,2-3,9-11H2,1H3,(H,21,25)
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| Chemical Name |
dithiolan-4-yl N-(2-butyl-1,3-dioxobenzo[de]isoquinolin-6-yl)carbamate
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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: 25 mg/mL (60.02 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 | 2.4009 mL | 12.0045 mL | 24.0090 mL | |
| 5 mM | 0.4802 mL | 2.4009 mL | 4.8018 mL | |
| 10 mM | 0.2401 mL | 1.2005 mL | 2.4009 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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