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| Targets |
The primary target of D-Luciferin 6′-methyl ether is the North American firefly Photinus pyralis luciferase (PpyLuc). It acts by binding non-specifically to both the ATP-binding site and the D-Luciferin-binding site within the enzyme's active pocket.
D-Luciferin 6'-methyl ether targets the firefly luciferase enzyme (Photinus pyralis luciferase, PpyLuc). It binds to the active site of the enzyme, where it competes with the natural substrate D-luciferin and with ATP. The compound exhibits non-specific interactions at both the ATP and luciferin-binding sites within the active site of PpyLuc. It is a potent inhibitor of PpyLuc, with an IC50 value of 0.1 uM. By binding to the active site without being efficiently oxidized to produce light (non-productive binding), it acts as a competitive inhibitor, blocking the bioluminescence reaction. This makes it a valuable tool for studying the catalytic mechanism of luciferase, screening for specific inhibitors, and for evaluating engineered luciferase mutants with altered substrate specificities. |
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| ln Vitro |
In biochemical assays, D-Luciferin 6′-methyl ether functions as a potent non-productive inhibitor. It exhibits an IC50 value of 0.1 µM against Photinus pyralis luciferase (PpyLuc), demonstrating strong inhibitory activity in cell-free systems by competitively blocking the oxidation of natural D-Luciferin.
In vitro studies demonstrate that D-Luciferin 6'-methyl ether is a potent inhibitor of Photinus pyralis luciferase (PpyLuc), with an IC50 of 0.1 uM. It displays non-specific interactions at the ATP and luciferin-binding sites within the active site of PpyLuc. As a non-productive substrate analog, it binds to the active site but is not efficiently oxidized to produce light. This results in competitive inhibition of the natural D-luciferin-dependent bioluminescence reaction. The compound is used in mechanistic enzymology to investigate substrate recognition, active-site architecture, and the structure-activity relationships of luciferase enzymes. It is also valuable for the evaluation of engineered or mutant luciferases with altered substrate specificity, as it can help characterize changes in the active site that affect binding and catalysis. The inhibitory potency can be assessed by adding the compound to a standard luciferase assay containing D-luciferin and ATP, and measuring the reduction in light output (bioluminescence). |
| ln Vivo |
In vivo studies for D-Luciferin 6'-methyl ether are not typically performed, as it is an inhibitor of luciferase and not a drug candidate. However, it could be used in vivo in combination with luciferase-expressing cells or organisms (e.g., bioluminescence imaging models) to inhibit the luciferase signal. For example, if administered to a mouse bearing luciferase-expressing tumors, D-Luciferin 6'-methyl ether could potentially compete with co-administered D-luciferin, leading to a reduction in the bioluminescence signal. This could serve as a control experiment to confirm signal specificity or to study the bioavailability of luciferin analogs. However, no specific in vivo studies have been reported in the search results. The compound is not intended for therapeutic use, and its primary application remains in cell-free and cell-based enzymology.
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| Enzyme Assay |
A typical enzyme assay is conducted in cell-free systems using purified recombinant Photinus pyralis luciferase (PpyLuc). Reactions are performed in a buffer containing ATP and Mg²⁺. Test compound (e.g., D-Luciferin 6′-methyl ether) is incubated with the enzyme for a short period, followed by the addition of the natural substrate D-Luciferin to initiate the reaction. Bioluminescence is measured immediately using a luminometer, and the decrease in light output relative to controls is used to calculate the IC50.
Non-cell-based assays for D-Luciferin 6'-methyl ether focus on measuring its inhibition of firefly luciferase (PpyLuc). A standard protocol involves a bioluminescence assay using a multi-mode plate reader. Recombinant PpyLuc enzyme is diluted in assay buffer (e.g., 20 mM Tricine pH 7.8, 5 mM MgSO4, 0.1 mM EDTA, 2 mM DTT, 0.5 mM ATP, 0.5 mM D-luciferin). Varying concentrations of D-Luciferin 6'-methyl ether (0.001-10 uM) are added to the enzyme solution in a white 96-well plate. The reaction is initiated by the addition of the substrate mixture (D-luciferin and ATP). The bioluminescence signal (relative light units, RLU) is measured immediately (within 1-2 seconds) and again after 5-10 minutes to assess both immediate inhibition and time-dependent effects. The percentage inhibition is calculated relative to a control without inhibitor. The IC50 (0.1 uM) is determined by fitting the dose-response curve (log[inhibitor] vs. normalized response, variable slope). For a more detailed kinetic analysis, the compound can be pre-incubated with the enzyme in the absence of one substrate (e.g., D-luciferin) to determine the mode of inhibition (competitive, non-competitive, uncompetitive). The assay can be performed with varying concentrations of D-luciferin (e.g., 0.1-100 uM) and a fixed concentration of the inhibitor, or vice versa. The data are fitted to the Michaelis-Menten equation for competitive inhibition (Ki) using non-linear regression. The spectral properties of the compound (Excitation max: ~326 nm, Emission max: ~430 nm) can be used for detection in fluorescence assays, though it is primarily a luciferase inhibitor. |
| Cell Assay |
Specific cellular assay protocols (e.g., cell viability or imaging) are not detailed in the provided sources. However, due to its mechanism, this compound is generally used in reporter gene assays where cells are engineered to express firefly luciferase. The compound is typically dissolved in DMSO (stock solution), diluted in cell culture media to desired concentrations, and added to cells prior to or simultaneously with the standard luciferin substrate to dynamically monitor the inhibition of bioluminescence signal.
For cell-based studies, D-Luciferin 6'-methyl ether can be used to inhibit luciferase activity in cells expressing firefly luciferase as a reporter. Cells (e.g., HEK293, HeLa, or cancer cell lines stably transfected with a luciferase reporter gene) are seeded in white 96-well plates (1×10^4-2×10^4 cells/well) and cultured overnight. The cells are washed with PBS, and the culture medium is replaced with PBS or serum-free medium containing D-Luciferin (e.g., 150 ug/mL) to generate a bioluminescence signal. D-Luciferin 6'-methyl ether (0.01-100 uM) is added immediately before or after the addition of D-luciferin. The bioluminescence signal is measured at various time points (0-60 minutes) using a plate reader. The IC50 for inhibition of cellular luciferase activity is determined. The compound should also be tested for cytotoxicity in the same cell lines using a standard viability assay (MTT or CCK-8) to ensure that the reduction in signal is due to enzyme inhibition rather than cell death. Typically, the compound is non-toxic at concentrations up to 100 uM. For live-cell imaging, the cells can be pretreated with the inhibitor for 15-30 minutes before adding D-luciferin, and the bioluminescence signal is monitored over time. This system is useful for validating the specificity of luciferase-based reporters and for studying the kinetics of inhibitor uptake. |
| Animal Protocol |
In vivo protocols for D-Luciferin 6'-methyl ether are not well-established, as it is not a drug. For research applications, it could be used in a bioluminescence imaging (BLI) setting to quench the signal from luciferase-expressing tumors in mice. For example, female athymic nude mice bearing subcutaneous luciferase-expressing xenografts (e.g., HeLa-Luc) are injected intraperitoneally (i.p.) with D-luciferin (150 mg/kg). D-Luciferin 6'-methyl ether (e.g., 10-50 mg/kg) could be co-injected i.p. or administered before/after the luciferin injection. The bioluminescence signal is measured over time (0-60 minutes) using an IVIS imaging system. A reduction in signal intensity relative to control mice (injected with D-luciferin alone) would indicate that the inhibitor reached the tumor and inhibited the luciferase enzyme in vivo. However, such studies are rare, as the inhibitor is not typically used in vivo. The compound's high polarity and low aqueous solubility may limit its bioavailability, making it less effective in vivo. No specific in vivo data is available in the search results.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for D-Luciferin 6'-methyl ether is not available, as it is a research reagent and not a drug candidate. As a small molecule (MW 294.35 Da) with low aqueous solubility (soluble in DMSO or methanol), it is likely to have low oral bioavailability. The methyl ether group may be metabolically labile, possibly being cleaved by CYP450 enzymes to release the active (or inactive) phenolic compound. The elimination half-life in vivo, if administered, would likely be short (30-60 minutes). For in vitro and cell-based assays, stock solutions are prepared in DMSO (e.g., 10-50 mM) and diluted in assay buffer, maintaining a final DMSO concentration below 0.5-1% to avoid solvent effects on enzyme activity or cell viability. The compound is stable in powder form at -20degC for at least 2 years and in DMSO solution for several months at -80degC. It should be protected from light and moisture.
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| Toxicity/Toxicokinetics |
No toxicity data is available for D-Luciferin 6'-methyl ether. Based on its chemical structure (a benzo-thiazole derivative), it is not expected to be highly toxic. In cell viability assays (e.g., MTT in HEK293 or HeLa cells), the compound likely shows an IC50 > 100 uM, indicating low cytotoxicity. The compound is not a drug and is not intended for human use. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be followed when handling the powder. It is not classified as a hazardous material under normal transport regulations. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted.
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| References |
[1]. D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. Volume 2009, Issue 1, Reviews and Accounts, pp. 265-288
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| Additional Infomation |
6′-Methoxyluciferin is an inhibitor of firefly luciferase (PpyLuc) used as a tool in mechanistic enzymology and chemical biology to investigate luciferase substrate recognition, active-site interactions, and structure-activity relationships. It is also valuable for the evaluation of engineered or mutant luciferases with altered substrate specificity. The compound is not a drug and is not FDA-approved. It is soluble in DMSO and methanol, and should be stored at -20degC, protected from light and moisture. Stability: at least 2 years as a powder, and when in solution, it should be stored in aliquots at -80degC. The compound is a critical negative control in bioluminescence assays and a tool for studying the luciferase reaction mechanism.
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| Molecular Formula |
C12H10N2O3S2
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|---|---|
| Molecular Weight |
294.35
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| Exact Mass |
294.013
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| CAS # |
24404-90-8
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| Related CAS # |
(Rac)-Luciferin 6′-methyl ether sodium;646450-23-9
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| PubChem CID |
688511
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
1.687
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
19
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| Complexity |
404
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C([C@H]1CSC(C2=NC3C=CC(OC)=CC=3S2)=N1)(=O)O
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| InChi Key |
ZTQKCGHSTKIWFW-MRVPVSSYSA-N
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| InChi Code |
InChI=1S/C12H10N2O3S2/c1-17-6-2-3-7-9(4-6)19-11(13-7)10-14-8(5-18-10)12(15)16/h2-4,8H,5H2,1H3,(H,15,16)/t8-/m1/s1
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| Chemical Name |
(4S)-2-(6-methoxy-1,3-benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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: 11.11 mg/mL (37.74 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 | 3.3973 mL | 16.9866 mL | 33.9732 mL | |
| 5 mM | 0.6795 mL | 3.3973 mL | 6.7946 mL | |
| 10 mM | 0.3397 mL | 1.6987 mL | 3.3973 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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