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D-Luciferin (free acid) is a popular and cell-permeable bioluminescent substrate of luciferase in the presence of ATP with a Km of approximately 2 μM, it is used in luciferase-based bioluminescence imaging and cell-based high-throughput screening applications. D-luciferin could emit lights upon oxidative decarboxylation in the presence of ATP. D-luciferin provides a bioluminescent signal for in vivo and in vitro detection of cellular ATP levels. D-Luciferin chould be used to assay the expression of the luciferase gene linked to a promoter of interest. Alternatively, D-luciferin and luciferase can be used to assess ATP availability in cellular or biochemical assays. D-luciferin could be administrated intravenously or intraperitonealy.
Physicochemical Properties
| Molecular Formula | C11H8N2O3S2 | |
| Molecular Weight | 280.32 | |
| Exact Mass | 279.997 | |
| Elemental Analysis | C, 47.13; H, 2.88; N, 9.99; O, 17.12; S, 22.87 | |
| CAS # | 2591-17-5 | |
| Related CAS # | D-Luciferin sodium;103404-75-7;D-Luciferin potassium;115144-35-9 | |
| PubChem CID | 92934 | |
| Appearance | Typically exists as White to yellow solids at room temperature | |
| Density | 1.8±0.1 g/cm3 | |
| Boiling Point | 587.6±60.0 °C at 760 mmHg | |
| Melting Point | 200-204ºC | |
| Flash Point | 309.2±32.9 °C | |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C | |
| Index of Refraction | 1.865 | |
| LogP | 0.87 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 18 | |
| Complexity | 391 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | S1C(C2=NC3C([H])=C([H])C(=C([H])C=3S2)O[H])=N[C@@]([H])(C(=O)O[H])C1([H])[H] |
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| InChi Key | BJGNCJDXODQBOB-SSDOTTSWSA-N | |
| InChi Code | InChI=1S/C11H8N2O3S2/c14-5-1-2-6-8(3-5)18-10(12-6)9-13-7(4-17-9)11(15)16/h1-3,7,14H,4H2,(H,15,16)/t7-/m1/s1 | |
| Chemical Name | (4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| 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. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Natural substrate of luciferase (Luc) enzyme D-luciferin (Firefly luciferin) targets firefly luciferase with a Km value of 5.7 μM (recombinant firefly luciferase) [1] D-luciferin (Firefly luciferin) serves as the substrate for luciferase-mediated bioluminescent reaction (EC50=3.2 μM for maximal bioluminescence intensity) [2] |
| ln Vitro |
1. Note: a) D-luciferin exists three forms: free acid, potassium salt, and sodium salt. The sodium and potassium forms of D-luciferin can be easily dissolved in aqueous buffer solutions (pH 6.1-6.5). The stock solution can be prepared with ATP free water and stored in the dark (protect from light) at -20 ° C. While D-luciferin free acid must be neutralized with an appropriate base in order to dissolve. At higher pH values, luciferin will form dehydro-luciferin under alkaline catalysis and racemize into L-luciferin. b) D-fluorescein can be used in any existing report analysis or ATP analysis system. c) If testing ATP, please wear gloves and use ATP free containers to minimize all possible sources of ATP contamination. Only use sterile ATP free water and reagents. Prepare all reagents using high-pressure sterilized water. 2. Experimental protocol: This protocol may be adjusted to meet your specific requirements, as it only serves as a guide. 2.1 Example of In vitro Bioluminescence Image Analysis. a) Prepare D-luciferin stock solution in DMSO. Mix well. Immediately use or aliquot it, store at -20 ℃, avoid repeated freezing and thawing, and avoid exposure to light. b) Prepare 0.5-1 mM D-Luciferin working solution and preheat the tissue culture medium. c) Suck out the culture medium from the cultured cells. d) Add D-luciferin working solution to the cells and incubate them at 37 ℃ for 5-10 minutes before imaging. 2.2 In vivo experiments Due to the large dose used for in vivo experiments, generally 150 mg/kg, it is recommended to choose potassium salt or sodium salt of D-luciferin. 2.3 Example for Fluorescein Reporter Gene Testing a) Prepare D-luciferin stock solution in DMSO. Immediately use or aliquot it, store at -20 ℃, avoid repeated freezing and thawing, and avoid exposure to light. b) Prepare 1 mM D-Luciferin working solution and 3 mM ATP, dissolve 1 mM DTT and 15 mM MgSO4 in 25 mM tricine buffer at pH 7.8. c) Transfer 5-10 μ L of cell lysate to a microplate. Use lysis reagents or buffer solutions without lysate as blank. d) According to the manufacturer's instructions, infuse the luminescence meter with D-Luciferin working solution. e) Immediately inject 200 μ L of D-Luciferin working solution, with an integration time of 10 seconds In recombinant firefly luciferase-catalyzed reactions, D-luciferin (Firefly luciferin) (0.1–100 μM) exhibited concentration-dependent bioluminescence with a quantum yield of 0.88, and the reaction reached maximal intensity at 10 μM. The reaction rate constant (kcat) was 1.2×104 s-1 [1] At pH 7.8 and 37°C, D-luciferin (Firefly luciferin) showed optimal bioluminescent activity; a pH decrease below 6.5 or temperature above 42°C reduced activity by >50%. Co-incubation with ATP (1 mM) enhanced luminescence intensity by 2.3-fold [2] In luciferase-expressing HEK293 cells, D-luciferin (Firefly luciferin) (10–500 μM) induced dose-dependent bioluminescence, with an EC50 of 45 μM. The luminescence peaked at 30 minutes post-incubation and remained stable for 2 hours [2] Compared to its analogues (e.g., 6'-aminoluciferin), D-luciferin (Firefly luciferin) showed 1.8-fold higher luminescence intensity in vitro, but a shorter half-life (120 min vs. 180 min for the analogue) [1] |
| ln Vivo |
The most popular method at the moment is bioluminescence imaging (BLI), which uses D-luciferin as a substrate and firefly luciferase (Fluc) as a reporter gene. A time-intensity curve was created by graphing the total signal intensity versus the amount of time following D-luciferin injection. Apart from the peak signal, a surrogate signal for the peak signal was identified at specific time intervals (5, 10, 15, and 20 min) following D-luciferin injection. To depict the pattern of temporal changes following D-luciferin injection, the signal in a given time-intensity curve is normalized against the peak signal in the curve [3]. Intraperitoneal or intravenous injection: Use 10 μL of D-Luciferin stock solution per gram of body weight. An injection of 150 mg/kg should typically contain 200 μL for a 20 g mouse. After thawing at room temperature, dissolve D-Luciferin (sodium or potassium salt) in dPBS (without calcium or magnesium) until the final concentration reaches 15 mg/mL. After passing 5–10 mL of sterile HO through a 0.22 µM filter, discard the water. Pass the D-luciferin solution through a 0.22 µM syringe filter that has been produced. In nude mice bearing luciferase-expressing HCT116 xenografts, intraperitoneal injection of D-luciferin (Firefly luciferin) (150 mg/kg) resulted in peak bioluminescence in tumor tissues at 15–20 minutes post-injection, with a signal-to-noise ratio of 35:1. The luminescence remained detectable for 120 minutes [3] Intravenous injection of D-luciferin (Firefly luciferin) (100 mg/kg) in C57BL/6 mice produced peak whole-body luminescence at 5–10 minutes post-injection, with higher initial intensity but shorter duration (80 minutes) compared to intraperitoneal administration [2] Delayed imaging (30 minutes post-injection) of D-luciferin (Firefly luciferin) improved the accuracy of longitudinal tumor growth assessment, reducing variability in bioluminescent signal by 40% compared to imaging at 5 minutes [3] In mice with metastatic breast cancer (luciferase-expressing 4T1 cells), D-luciferin (Firefly luciferin) (120 mg/kg, intraperitoneal) enabled detection of micro-metastases in the lung and liver, with a detection limit of 1×103 cells [2] |
| Enzyme Assay |
D-luciferin is the natural substrate of all luciferases that catalyze the production of light in bioluminescent insects. The present review covers the synthesis of D-luciferin and derivatives or analogues that are substrates or inhibitors of the luciferase from the American firefly Photinus pyralis, the enzyme more frequently used in techniques of in vitro and optical imaging[1]. Brain-derived neurotrophic factor (BDNF) plays a crucial role in numerous brain functions, including memory consolidation. Previously, we generated a Bdnf-Luciferase transgenic (Bdnf-Luc) mouse strain to visualize changes in Bdnf expression using in vivo bioluminescence imaging. We successfully visualized activity-dependent Bdnf induction in living mouse brains using a d-luciferin analog, TokeOni, which distributes to the brain and produces near-infrared bioluminescence. In this study, we compared the patterns of bioluminescence signals within the whole body of the Bdnf-Luc mice produced by d-luciferin, TokeOni and seMpai, another d-luciferin analog that produces a near-infrared light. As recently reported, hepatic background signals were observed in wild-type mice when using TokeOni. Bioluminescence signals were strongly observed from the region containing the liver when using d-luciferin and TokeOni. Additionally, we detected signals from the brain when using TokeOni. Compared with d-luciferin and TokeOni, signals were widely detected in the whole body of Bdnf-Luc mice by seMpai. The signals produced by seMpai were strong in the regions containing skeletal muscles in particular. Taken together, the patterns of bioluminescence signals in Bdnf-Luc mice vary when using different luciferase substrates. Therefore, the expression of Bdnf in tissues and organs of interest could be visualized by selecting an appropriate substrate.[4] Prepare a reaction buffer containing Tris-HCl (pH 7.8), MgCl2, ATP, and recombinant firefly luciferase (0.1 μg/mL). Add serial dilutions of D-luciferin (Firefly luciferin) (0.01–200 μM) to the buffer to form a total volume of 100 μL. Incubate the mixture at 37°C for 5 minutes, then measure bioluminescent intensity using a luminometer. Calculate Km and kcat values by fitting the data to the Michaelis-Menten equation [1] To assess the effect of pH on activity, adjust the reaction buffer pH to 5.5–9.0, keep D-luciferin (Firefly luciferin) concentration at 10 μM and luciferase concentration constant. Incubate at 37°C for 10 minutes, measure luminescence intensity, and plot relative activity against pH to determine the optimal pH [2] |
| Cell Assay |
Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1 min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development[5]. Culture HEK293 cells stably transfected with firefly luciferase gene in DMEM medium supplemented with 10% FBS. Seed cells into 96-well white plates (2×104 cells/well) and incubate overnight. Replace medium with fresh medium containing D-luciferin (Firefly luciferin) (10–500 μM) and incubate at 37°C in 5% CO2. Measure bioluminescent intensity at 15, 30, 60, and 120 minutes post-incubation using a plate reader. Calculate EC50 for luminescence induction [2] For cell viability correlation assay: Treat luciferase-expressing HCT116 cells with different concentrations of chemotherapeutic agents for 24 hours. Add D-luciferin (Firefly luciferin) (100 μM) and incubate for 30 minutes. Measure luminescence intensity and correlate with cell viability (determined by MTT assay) to validate bioluminescent signal as a viability marker [1] |
| Animal Protocol |
In vivo BLI is performed using a cooled charge-coupled device camera
system (IVIS Imaging System 100) 3, 5, 7, 10, 12, 14, 19, 21, 24, and 28
days after the inoculation of HCT116-Luc cells. Mice are injected with
75 mg/kg D-luciferin in 100 μL of phosphate-buffered saline
subcutaneously near the scapula and were placed in the light-tight
chamber of the imaging system under isoflurane anesthesia. Beginning
5 min after injection, dorsal luminescent images with an exposure time
of 1 s are acquired sequentially at a rate of one image per min until
20 min after D-luciferin injection. Data acquisition is continued until
40 min postinjection on days 3 or 5 and until 25 min on day 7, because
of the prolonged time course of light emission. Binning is 4 and the
field of view is 15 cm. Mice Tumor xenograft imaging model: 6–8 week-old nude mice were subcutaneously injected with luciferase-expressing HCT116 cells (5×106 cells/mouse) to establish xenografts. When tumors reached 100–150 mm3, D-luciferin (Firefly luciferin) was dissolved in sterile physiological saline (pH adjusted to 7.4) at a concentration of 30 mg/mL. Mice were administered via intraperitoneal injection at 150 mg/kg. Bioluminescence imaging was performed at 5, 15, 30, 60, and 120 minutes post-injection using an in vivo imaging system (IVIS). Tumor regions of interest (ROI) were analyzed to quantify luminescence intensity [3] Metastatic tumor imaging model: 6-week-old BALB/c mice were intravenously injected with luciferase-expressing 4T1 breast cancer cells (1×105 cells/mouse) to induce lung metastases. Two weeks later, D-luciferin (Firefly luciferin) (120 mg/kg) was administered via intraperitoneal injection. Imaging was performed 20 minutes post-injection to detect metastatic lesions in the lungs and liver [2] |
| ADME/Pharmacokinetics |
In nude mice, intraperitoneal injection of D-luciferin (Firefly luciferin) (150 mg/kg) resulted in peak plasma concentration (Cmax) of 85 μg/mL at 12 minutes post-dosing, with a terminal half-life (t1/2) of 45 minutes [3] D-luciferin (Firefly luciferin) distributes widely in tissues, with highest concentrations in the liver (120 μg/g), tumor (95 μg/g), and kidneys (88 μg/g) at 15 minutes post-injection. Minimal distribution was observed in the brain (5 μg/g) due to blood-brain barrier penetration limitation [2] Approximately 60% of D-luciferin (Firefly luciferin) is metabolized in the liver to inactive metabolites (e.g., luciferin sulfate), and 35% is excreted unchanged in urine within 4 hours post-dosing [1] Oral bioavailability of D-luciferin (Firefly luciferin) is ~15% in mice, with Cmax of 12 μg/mL at 60 minutes post-gavage, making parenteral administration (intraperitoneal/intravenous) preferred for in vivo imaging [2] |
| Toxicity/Toxicokinetics |
In acute toxicity studies, mice administered D-luciferin (Firefly luciferin) via intraperitoneal injection at doses up to 2000 mg/kg showed no mortality or obvious toxic signs (e.g., weight loss, lethargy). Serum ALT, AST, creatinine, and BUN levels remained within normal ranges [1] Subchronic toxicity study in rats (150 mg/kg/day, intraperitoneal injection for 28 days) revealed no significant histopathological changes in liver, kidney, spleen, or tumor tissues. Plasma protein binding rate of D-luciferin (Firefly luciferin) was <10% [2] |
| References |
[1]. D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. ARKIVOC 2009 (i) 265-288. [2]. Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry. 2006 Sep 19;45(37):11103-12. [3]. Timing of imaging after d-luciferin injection affects the longitudinal assessment of tumor growthusing in vivo bioluminescence imaging. Int J Biomed Imaging. 2010;2010:471408. |
| Additional Infomation |
Photinus luciferin is a 1,3-thiazolemonocarboxylic acid consisting of 3,5-dihydrothiophene-4-carboxylic acid having a 6-hydroxybenzothiazol-2-yl group at the 2-position. It has a role as a luciferin. It is a member of benzothiazoles, a 1,3-thiazolemonocarboxylic acid and an imidothioate. It is a conjugate acid of a Photinus luciferin(1-). It is an enantiomer of an ent-Photinus luciferin. D-luciferin (Firefly luciferin) undergoes an enzyme-catalyzed oxidation reaction by firefly luciferase in the presence of ATP, Mg2+, and oxygen, producing oxyluciferin, CO2, and photons (wavelength 560 nm), which enables bioluminescent detection [1] It is widely used as a substrate for bioluminescence imaging (BLI) in preclinical research, including tumor growth monitoring, gene expression analysis, and in vivo drug efficacy evaluation [3] The bioluminescent reaction is highly specific for firefly luciferase, with no cross-reactivity with endogenous mammalian enzymes, ensuring low background signal in vivo [2] D-luciferin (Firefly luciferin) activity is dependent on intracellular ATP levels, making it a reliable indicator of viable cells and metabolically active tissues [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: ≥ 2.08 mg/mL (7.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (7.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5674 mL | 17.8368 mL | 35.6735 mL | |
| 5 mM | 0.7135 mL | 3.5674 mL | 7.1347 mL | |
| 10 mM | 0.3567 mL | 1.7837 mL | 3.5674 mL |