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Purity: ≥98%
Diphenylterazine (DTZ) is a novel bioluminescence agent that showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. As a Red-shifted bioluminescence reporter, it has the potential to be used for biological imaging.
| Targets |
Bioluminescence agent
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|---|---|
| ln Vitro |
Millimolar concentrations of diphenylterazine have negligible cytotoxic effects [1]. Diphenyltrazine has strong in vivo pharmacokinetics, red-shifted emission, a high quantum yield, and the absence of cofactors needed for light emission. Yeh AH (2023) designed a small and stable protein scaffold from scratch to make the size and shape of the pocket suitable for diphenyltetrazine using the multinuclear transport factor NTF2-like superfamily as the target topology and diphenyltrazine as the target substrate of luciferase. By removing luciferase with a high degree of selectivity, this technique overcomes the limitations of naturally occurring proteins. Although the substrate apex of the de novo designed luciferase is higher, its catalytic efficiency towards diphenyltetrazine (kcat/Km = 106/M/s) is similar to that of natural luciferase [2]. NOTE: To make a 30 mM DTZ stock solution with 5 mM L-ascorbic acid, dissolve 1 mg of DTZ in 88 μL of master mix after first making a premix by dissolving 17.6 mg of L-ascorbic acid in 10 mL of ethanol and 10 mL of 1,2-propanediol [3].
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| ln Vivo |
Background emission is absent when diphenyltetrazine is injected into untransfected BALB/c electrodes. Extended kinetics are shown in the bioluminescence generated by intraperitoneal injection of diphenyltetrazine [1]. The xenograft NU/J tumor model can be used to track the growth of treatment with diphenyltetrazine (0.3 μMol/mouse; 1.13 mg.mL–1/100 μL/mouse) [4].
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| Enzyme Assay |
De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence-structure relationships. Here we describe a deep-learning-based 'family-wide hallucination' approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M-1 s-1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.[2]
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| Cell Assay |
Staining examples:
Example 1: Diphenylterazine may be used as a substrate for Antares2. Method: For labeling of cells. 1. Add Diphenylterazine (500, 50, 5, and 0.5 μM) into the culture media containing PC3/CD63-Antares2 cells. 2. Image with a bioluminescence imaging system. Example 2: Diphenylterazine (DTZ) may be used as a bioluminescence agent for bioluminescent imaging in vitro and in vivo. Method: For in vivo usage. 1. Anesthetized animals. 2. Animals are anesthetized again after the first was anesthetized for 36 hours and add saline containing Diphenylterazine to the eye surface of animals (for eye studies). After being anesthetized for 48 hours, inject Diphenylterazine solution into both the left and the right legs by i.p./intraperitoneal injection (for skeletal muscle studies). Example 3: Diphenylterazine may be used to track tumor growth in vivo. Method: For in vivo usage. 1. Diphenylterazine (0.3 μM) is injected intravenously into testing animals. 2. Image with an in vivo imaging system for image. |
| Animal Protocol |
Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivo applications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shifted emission and improved water solubility. Through directed evolution, we engineered a LumiLuc luciferase with broad substrate specificity. In the presence of corresponding pyridyl substrates (i.e., pyCTZ, 6pyDTZ, or 8pyDTZ), LumiLuc generates highly bright blue, teal, or yellow bioluminescence. We compared our LumiLuc-8pyDTZ pair with several benchmark reporters in a tumor xenograft mouse model. Our new pair, which does not need organic cosolvents for in vivo administration, surpasses other reporters by detecting early tumors. We further fused LumiLuc to a red fluorescent protein, resulting in a LumiScarlet reporter with further red-shifted emission and enhanced tissue penetration. LumiScarlet-8pyDTZ was comparable to Akaluc-AkaLumine, the brightest ATP-dependent luciferase-luciferin pair, for detecting cells in deep tissues of mice. In summary, we have engineered a new family of ATP-independent bioluminescent reporters, which will have broad applications because of their ATP-independency, excellent biocompatibility, and superior in vivo sensitivity.[4]
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| References | |
| Additional Infomation |
Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.[1]
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| Molecular Formula |
C25H19N3O
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|---|---|
| Molecular Weight |
377.437865495682
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| Exact Mass |
377.152
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| Elemental Analysis |
C, 79.55; H, 5.07; N, 11.13; O, 4.24
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| CAS # |
344940-63-2
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| PubChem CID |
135439143
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| Appearance |
Orange to reddish brown solid powder
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| LogP |
6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
510
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
HYQVAZNNCBIZSK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H19N3O/c29-25-21(16-18-10-4-1-5-11-18)27-24-23(20-14-8-3-9-15-20)26-22(17-28(24)25)19-12-6-2-7-13-19/h1-15,17,26H,16H2
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| Chemical Name |
2-benzyl-6,8-diphenylimidazo[1,2-a]pyrazin-3(7H)-one
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| Synonyms |
DTZ; 2-benzyl-6,8-diphenylimidazo[1,2-a]pyrazin-3-ol; 2-Benzyl-6,8-diphenylimidazo[1,2-a]pyrazin-3(7H)-one; DTZ; Diphenylterazine (DTZ)?; 2-benzyl-6,8-diphenyl-7H-imidazo[1,2-a]pyrazin-3-one; SCHEMBL19912656;
Diphenylterazine
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
Solubility in DMF : ~11.0 mg/mL (~29.0 mM, with sonication; Note: DMSO can inactivate the activity of Diphenylterazine)
Solubility in EtOH+HCl : ~1 mg/mL (~2.6 mM; with sonication and warming, and adjust pH to 2 with 1M HCl and heat to 80°C;Note: DMSO can inactivate the activity of Diphenylterazine) Solubility in H2O : Insoluble (< 0.1 mg/mL; Note: DMSO can inactivate the activity of Diphenylterazine) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.11 mg/mL (2.94 mM) (saturation unknown) in 10% DMF 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2 mg/mL (5.30 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O 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 | 2.6494 mL | 13.2471 mL | 26.4943 mL | |
| 5 mM | 0.5299 mL | 2.6494 mL | 5.2989 mL | |
| 10 mM | 0.2649 mL | 1.3247 mL | 2.6494 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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Reference:Nat Methods. 2017 Oct; 14(10): 971–974. |
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