Natural substrate of luciferase (Luc) enzyme

1. Note:
a) The D-luciferin salts (sodium or potassium) exhibits a high degree of solubility, up to 100 mM, in aqueous buffer (pH 6.1-6.5). The stock solution can be prepared with ATP free water and stored in the dark at -20 ° C. Free acids must be neutralized with an appropriate base in order to dissolve. At higher pH values, fluorescein will form dehydrofluorescein under alkaline catalysis and racemize into L-isomer (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.
The following scheme is an example of preparation of D-Luciferin sodium/potassium salts. It is suitable for most cell types and in vivo animal use.
2.1 Example of In vitro Bioluminescence Image Analysis.
a) Prepare 100 mM (100-200X) D-fluorescein sodium or potassium stock solution in sterile water. Mix well. Immediately use or aliquot it, store at -20 ° C, avoid freeze-thaw cycles, and avoid exposure to light.
b) Prepare 0.5-1 mM D-Luciferin working solution in pre-heated 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 ° C for 5-10 minutes before imaging.
2.2 Example for analyzing bioluminescence images in vivo
a) Prepare 15 mg/mL D-Luciferin stock solution in DPBS, free of Mg2+and Ca2+. Mix evenly.
b) The filter removes bacteria from the solution through a 0.2 μ M filter. Immediately use or aliquot it and store at -20 ° C to avoid freeze-thaw cycles and exposure to light.
c) 10-15 minutes before imaging, intraperitoneal (IP) injection of D-Luciferin at 150 mg/kg (or 10 μ L/g fluorescein stock solution) of animal body weight.
Attention: Fluorescein kinetics studies should be conducted on each animal model to determine peak signal time.
2.3 Example for D-Luciferin Reporter Gene Testing
a) Prepare 100 mM D-Luciferin stock solution in sterile water. Immediately use or aliquot it, store at -20 ° C, avoid freeze-thaw cycles, and avoid exposure to light.
b) Prepare 1 mM D-Luciferin working solution and 3 mM ATP, 1 mM DTT, and 15 mM MgSO4 in 25 mM Tricine buffer 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-Luciferinworking solution.
e) Immediately inject 200 μ L of D-Luciferin working solution, with an integration time of 10 seconds.

The most popular method at the moment is bioluminescence (BLI), which uses D-luciferin substrate and firefly luciferase (Fluc) as a reporter gene. A time-intensity curve was created by graphing the overall signal intensity versus the amount of time following D-luciferin injection. Apart from the peak signal, surrogate signals for the peak signal were identified as the signals at predetermined 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]. Use 10 μL of D-luciferin stock solution (intraperitoneal or intravenous) for every gram of body weight. An injection of 20 g should typically contain 200 μL due to the conventional dose of 150 mg/kg. To dissolve the D-luciferin (potassium or sodium salt) solution to a final concentration of 15 mg/mL, thaw it and dilute it in dPBS (clear calcium or magnesium). Wet a 0.22 µM filter with 5–10 mL of sterile HO, then drain. ..Pass the D-luciferin solution through a 0.22 µM syringe filter that has been produced.

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].

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring.[2]

[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.

The fascinating phenomenon of bioluminescence and its wide range of biotechnological applications in optical imaging make the chemical properties of D-luciferin and its analogues extremely valuable for research. This surpasses the early expectations put forward by White et al. in 1971: "The excited states of chemical synthesis play a crucial role in bioluminescence; they will also play an important role in other areas of biology."¹ The synthetic methods of related benzothiazole systems have not changed much compared to the original research nearly forty years ago. However, the chemical principles remain reliable and are used to prepare D-luciferin and related compounds. These compounds can serve as substrates for luciferases and be applied in applications such as optical imaging technology, which is now widely used for preclinical molecular imaging of cells and small animals. The classic enzyme PpyLuc, used for this purpose, emits a yellow-green light with a broad emission spectrum, a maximum emission wavelength of 560 nm, and low background bioluminescence. This makes in vivo bioluminescence imaging using PpyLuc a simple and highly sensitive method for small animal molecular imaging. Regulating the wavelength of emitted light is an important research goal, currently mainly achieved through site-directed mutagenesis of luciferase amino acid residues. Although similar effects may be achieved by chemically modifying the structure of D-luciferin, there are not many modifications that can be made for such a relatively simple structure as D-luciferin. In fact, only a few compounds have been synthesized so far, and only a few compounds have been shown to redshift PpyLuc-catalyzed bioluminescence. However, the examples listed in this article show that, in addition to biochemical modification of enzymes, clever ideas and innovative applications of synthetic organic chemistry can also bring new breakthroughs to this fascinating research field. Finally, other analogues of D-luciferin may meet the expectations for new applications in in vivo optical imaging, as recent reports have revealed, which have elucidated the possibility of binding various compounds (including peptides or functionalized polyethylene glycols) to the amino group of D-6′-aminoluciferin. [1]

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Staining Example 1:
Sodium D-luciferin can be used as a substrate for luciferase in vivo imaging.
Method: For bioluminescence imaging.
1) Mice were anesthetized and then injected with sodium D-luciferin (75 mg/kg) for imaging.
2) Imaging was performed using a bioluminescence imaging system. Staining Example 2: D-fluorescein sodium can be used as a substrate for luciferase in in vivo imaging to monitor tumor growth. Method: For bioluminescent imaging. 1) Inject D-fluorescein sodium (150 mg/kg; intraperitoneal injection) into mice. 2) Image using a bioluminescent imaging system. Staining Example 3: D-fluorescein sodium can be used as a substrate for luciferase (LUC) detection. Method: For luciferase (LUC) detection. 1) Incubate plant samples (leaves) with D-fluorescein sodium (1 mM; 10 min) and fluorescein. 2) Acquire signals and images using a Photek camera. Staining Example 4: D-fluorescein sodium can be used as a substrate for luciferase in in vivo imaging to monitor tumor growth. Method: For bioluminescent imaging.
1) D-fluorescein sodium (150 mg/kg) was injected intraperitoneally into mice.
2) Bioluminescence imaging was performed using the IVIS Lumina XRMS series.

C11H7N2NAO3S2

302.29

301.979

C, 43.71; H, 2.33; N, 9.27; Na, 7.61; O, 15.88; S, 21.21

103404-75-7

D-Luciferin;2591-17-5;D-Luciferin potassium;115144-35-9

2733762

Typically exists as Light yellow to yellow solids at room temperature

473.7ºC at 760mmHg

240.3ºC

2.78E-10mmHg at 25°C

0.049

1

7

2

19

396

1

S1C(C2=NC3C([H])=C([H])C(=C([H])C=3S2)O[H])=N[C@@]([H])(C(=O)[O-])C1([H])[H].[Na+]

BZNVUYVALNTPBG-WJCSTRGMSA-M

InChI=1S/C11H8N2O3S2.Na/c14-5-1-2-6-8(3-5)18-10(12-6)9-13-7(4-17-9)11(15)16/h1-3,7,13H,4H2,(H,15,16)/q+1/p-1/b10-9+/t7-/m1./s1

sodium (S,E)-2-(6-oxobenzo[d]thiazol-2(6H)-ylidene)thiazolidine-4-carboxylate

D-Luciferin Sodium; D-Luciferin sodium salt; Sodium (S)-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate; D-Luciferin Sodium; D-Luciferin, Sodium Salt; D-Luciferin (sodium); C11H7N2NaO3S2; D-Luciferin sodium salt monohydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~250 mg/mL (~826.99 mM)
DMSO : ~100 mg/mL (~330.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.27 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 25.0 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.

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Solubility in Formulation 3: 100 mg/mL (330.80 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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