Bioluminescence agent
Luciferase variants (teLuc, Antares2, ATP-independent luciferase variants) [1][3][4]

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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- Diphenylterazine (DTZ) functions as a luciferin substrate, forming bioluminescent complexes with various luciferase variants. When paired with red-shifted luciferases (e.g., teLuc, Antares2), it emits red-shifted bioluminescence with a peak emission wavelength of ~620-640 nm [1][3][4]
- In vitro bioluminescence assays showed that DTZ exhibited higher light output and longer luminescence duration when combined with ATP-independent luciferase variants compared to ATP-dependent counterparts (detected by microplate reader). The signal-to-noise ratio was increased by ~3-5 fold [4]
- DTZ demonstrated good stability in vitro buffer systems (pH 7.2-7.4) at 37°C, with no significant degradation within 2 hours [3]
- When incubated with luciferase-expressing cells (e.g., HEK293T cells transfected with Antares2 or teLuc plasmid), DTZ (10-50 μM) induced strong, stable bioluminescent signals that were detectable for up to 4 hours (detected by cell imaging system) [1][3]

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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- In nude mice bearing luciferase-expressing tumor xenografts (HEK293T-Antares2), intraperitoneal injection of DTZ (10 mg/kg) produced strong red-shifted bioluminescent signals in tumor tissues, with peak signal at 15-30 minutes post-administration. The signal was detectable for ~6 hours, enabling long-term in vivo imaging [1][3]
- DTZ-mediated bioluminescence showed enhanced tissue penetration in mice compared to traditional luciferin substrates (e.g., D-luciferin), allowing clear visualization of deep-seated tumors and internal organs [1][4]
- In mice injected with ATP-independent luciferase-expressing cells, DTZ (10 mg/kg, intravenous injection) maintained stable bioluminescent signals under hypoxic conditions (e.g., tumor microenvironment), whereas ATP-dependent substrate signals were significantly reduced [4]

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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- Luciferase activity assay (in vitro): Recombinant luciferase variants (teLuc, Antares2, ATP-independent variants) were diluted in reaction buffer (pH 7.4). DTZ was added to the enzyme solution at final concentrations of 1-100 μM, and bioluminescence intensity and duration were measured immediately using a luminometer. Kinetic parameters (e.g., luminescence half-life, maximum light output) were calculated from the luminescence kinetic curves [1][4]
- Substrate specificity assay: DTZ (50 μM) was incubated with different luciferase families (firefly luciferase, Renilla luciferase, red-shifted variants) in parallel. Bioluminescence signals were detected to evaluate the selective binding of DTZ to red-shifted and ATP-independent variants [1][4]

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.

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- Luciferase-expressing cell preparation: HEK293T or other cell lines were transfected with luciferase (teLuc/Antares2/ATP-independent variant) expression plasmids using transfection reagents. Transfected cells were cultured for 24-48 hours to ensure sufficient luciferase expression [1][3][4]
- In vitro cell imaging assay: Transfected cells were seeded in 96-well plates or imaging dishes. DTZ was added to the cell culture medium at final concentrations of 10-50 μM. Bioluminescent signals were captured at different time points (0-4 hours) using a cell bioluminescence imaging system. Signal intensity and duration were quantified using image analysis software [1][3]
- Hypoxic cell assay: Luciferase-expressing cells were cultured in hypoxic chambers (1% O₂) for 12 hours. DTZ (20 μM) was added, and bioluminescent signals were measured and compared with normoxic controls [4]

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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- Tumor xenograft imaging model: Nude mice were subcutaneously injected with luciferase-expressing HEK293T cells (5×10⁶ cells/mouse) to form xenografts. When tumors reached ~500 mm³, mice were randomly divided into experimental and control groups [1][3]
- DTZ preparation and administration: DTZ was dissolved in DMSO (10% v/v) and diluted with sterile PBS or normal saline to the desired concentration. The experimental group received DTZ via intraperitoneal (10 mg/kg) or intravenous (5-10 mg/kg) injection; the control group received vehicle (DMSO/PBS) [1][3][4]
- In vivo imaging: Mice were anesthetized with isoflurane, and bioluminescent signals were captured at 5, 15, 30, 60, 120, 360 minutes post-administration using an in vivo imaging system. Signal intensity in target tissues (tumor, organs) was quantified using imaging software [1][3][4]
- Toxicity observation: Mice were monitored for body weight, food intake, and general behavior for 7 days post-DTZ administration. No obvious abnormalities were recorded [3][4]

In in vivo experiments, doses up to 10 mg/kg (intraperitoneal/intravenous injection) of DTZ did not cause significant changes in mouse body weight, organ coefficients (liver, kidney, spleen), or serum biochemical indicators (ALT, AST, Cr, BUN), indicating low acute toxicity [3][4]. In in vitro experiments, concentrations up to 50 μM of DTZ did not affect the viability of HEK293T cells (as detected by the MTT assay) [3].

[1]. Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging. Nat Methods. 2017 Oct;14(10):971-974.

[2]. De novo design of luciferases using deep learning. Nature. 2023 Feb;614(7949):774-780.

[3]. Practical Notes for teLuc-DTZ and Antares2-DTZ (updated 07/29/2019).

[4]. ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging. ACS Chem Biol. 2019 May 17;14(5):959-965.

Redshift bioluminescent reporter molecules have important applications in the field of bioimaging. We describe the development of redshift luciferins based on synthetic coelentrin analogs and their corresponding NanoLuc mutants, which enable bright bioluminescence. One pair of compounds showed superior sensitivity to commonly used bioluminescent reporter molecules both in vitro and in vivo. Based on this pair of compounds, we developed an Antares reporter molecule, Antares2, based on bioluminescent resonance energy, which provides better signals from deep tissues. [1]

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- Diphenyltetraazine (DTZ) is a synthetic small molecule luciferin substrate designed for redshift bioluminescent imaging. [1][4]
- Its core mechanism involves a bioluminescent reaction with a luciferase variant: luciferase catalyzes the oxidation of DTZ to produce excited-state intermediates that emit redshifted light when returning to the ground state. For certain luciferase variants, the reaction is ATP-independent[4] - DTZ has advantages such as redshift of emission spectrum (enhanced tissue penetration), long luminescence duration, good stability, and low toxicity, making it suitable for in vitro and in vivo bioluminescence imaging of tumors, organs, and biological processes[1][3][4] - It is mainly used as a research tool for molecular biology, preclinical imaging, and biomedical research, rather than as a therapeutic drug[1][2][3][4]

C25H19N3O

377.437865495682

377.152

C, 79.55; H, 5.07; N, 11.13; O, 4.24

344940-63-2

135439143

Orange to reddish brown solid powder

6

1

3

4

29

510

0

HYQVAZNNCBIZSK-UHFFFAOYSA-N

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

2-benzyl-6,8-diphenylimidazo[1,2-a]pyrazin-3(7H)-one

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

2934.99.9001

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.

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

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 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.

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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.

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