Luminescent enzyme substrate

Bioluminescence-based techniques, such as bioluminescence imaging, BRET and dual-luciferase reporter assay systems, have been widely used to examine a myriad of biological processes. Coelenterazine (CTZ), a luciferin or light-producing compound found in bioluminescent organisms, has sparked great curiosity and interest in searching for analogues with improved photochemical properties. This review summarizes the current development of coelenterazine analogues, their bioluminescence properties, and the rational design of caged coelenterazine towards biotargets, as well as their applications in bioassays. It should be emphasized that the design of caged luciferins can provide valuable insight into detailed molecular processes in organisms and will be a trend in the development of bioluminescent molecules.[1]

Caged-coelenterazine derivatives as bioluminescent probes for investigating enzyme activity. Bioluminescence imaging (BLI) is becoming an important technique that is readily applied in the life sciences. The coelenterate-luciferase system, which does not need extra co-factors in addition to molecular oxygen, is simpler than the firefly luciferase system or bacterial system. Therefore, coelenterazine derivatives could act as bioluminescent probes to monitor and image events in vitro and in vivo. In 2013, the Kazuya Kikuchi lab reported two caged coelenterazine derivatives, 132 and 133 (Fig. 11). They were designed and synthesized for imaging of β-galactosidase activity and expression in HEK-293T cell cultures that expressed a mutant Gaussia luciferase. These compounds were introduced with β-galactosidase cleavable caging groups at the key site of bioluminescence, the carbonyl group of the imidazopyrazinone moiety, which resulted in low auto-oxidation of coelenterazine and high specificity for β-galactosidase. The caged coelenterazine probes themselves did not emit bioluminescence with luciferase. However, in the presence of β-galactosidase, the probes containing β-galactosidase cleavable groups were cleaved by enzymes and free coelenterazine was generated to react with Gaussia luciferase to produce a bioluminescent signal (Fig. 11). Furthermore, the free coelenterazine could be generated from compound 133 in β-Gal-expressing cells. Also, it would only generate bioluminescence when it readily diffused to GLucM23-expressing cells with outer membrane bound Gluc or intracellularly localized Gluc (Fig. 11). In other words, the probes only induce bioluminescence when they are cleaved by β-galactosidase and encounter luciferase. Therefore, compound 133 has the potential to be used as a dual reporter in two different cell populations.83 These were the first probes based on a caged coelenterazine strategy to evaluate enzyme activity successfully. Their study can be helpful for further research on bioluminescent probes based on coelenterazine and their applications. It is difficult to introduce a large caging group at the C-3 position of the imidazopyrazinone core. Hence, this is an excellent example to guide the design of caged coelenterazine-type probes.[1]

Coelenterazine-type bioluminescent probes for monitoring cell membrane fusion In 2013, the Kikuchi lab reported the first membrane impermeable coelenterazine derivative (Fig. 13), compound 145, which had the potential to be used as a bioluminescent probe for the monitoring of cell membrane fusion events (Fig. 14).85 This compound was synthesized by alkylating the coelenterazine with a linker containing a terminal anionic phosphonate moiety. Meanwhile, two other coelenterazine derivatives (143 and 144) containing a polyethylene glycol (PEG) linker with terminal benzyl-protecting groups were synthesized to explore the effect on bioluminescence. However, their performance was not as good as that of probe 145. Probe 145 was highly cell-impermeable due to its negative charge, resulting in failure to penetrate the cell membrane to react with intracellularly localized GlucER. However, probe 145 could emit a bioluminescence signal with outer-membrane bound GlucM23Mem. Also, if the secretory vesicle carrying Gluc fused with cell membranes, Gluc would be exposed to probe 145, thereby leading to the generation of bioluminescence. In addition, it is interesting that compound 145 showed a 30 fold higher bioluminescence activity for Gaussia luciferase (Gluc) over Renilla luciferase (RLuc), which indicated that Gluc was more appropriate. This probe is an example of the use of small molecular probes to explore biological processes, considering the lower application of coelenterazine-type probes.[1]

[1]. Lighting up bioluminescence with coelenterazine: strategies and applications. Photochem Photobiol Sci. 2016;15(4):466-480.

The development of coelentrins has focused on two main directions. One strategy involves conventional modification of coelentrin substrates, particularly optimizing the substituents at the C-2, C-6, and C-8 positions of the imidazopyrazinone core. Over the past two decades, significant work has been conducted to discover more suitable compounds to enhance luminescence intensity, achieve redshift emission, and improve bioluminescence stability. Generally, introducing electron-donating groups at the C-2, C-6, and C-8 positions, increasing molecular polarity or conjugation, has a significant effect on improving bioluminescence intensity or influencing the position of the maximum emission peak. However, only a few compounds exhibit excellent performance across all aspects, enabling their application in bioluminescence detection and replacement of natural coelentrins. Another strategy involves encasing the 3-carbonyl site of the coelentrin imidazopyrazinone skeleton, a key active site for bioluminescent reactions. Highly stable cage-like coelentrin derivatives only emit bioluminescence under specific conditions. Therefore, cage-like coelentrin derivatives can be used as sustained-release substrates or bioluminescent probes for the detection of biomacromolecules and bioactive small molecules. Recently, several cage-like firefly luciferins have been reported as bioluminescent probes, some exhibiting excellent performance (sup>4,88–95). However, research on cage-like coelenterin is scarce. The limited application is likely due to the extremely low reported yields of cage-like reactions. This may be because the 3-carbonyl group of the imidazopyrazinone ring is sensitive to bases and oxygen, making cage-like modification difficult. Furthermore, coelenterin and its derivatives appear to be less stable than firefly luciferins, which may be a major obstacle to their application. Another drawback of coelenterin is its maximum emission wavelength of less than 600 nm, making it unsuitable for deep tissue imaging. Nevertheless, coelenterin bioluminescent systems still possess significant advantages. This versatile luciferin can be used in various bioluminescent luciferase systems, such as Renilla luciferase and Gaussian luciferase. Moreover, most coelenterin bioluminescent systems consist only of luciferin and luciferase, without other components, resulting in a simpler structure. Currently, luciferin-luciferase bioluminescence technology is widely used in various fields of chemical biology. It is worth emphasizing that the design of cage-like luciferin can provide valuable insights into the molecular processes of organisms and represents a new trend in the development of bioluminescent molecules. Cage-like coelenterate probes, as an important class of bioluminescent molecules, are expected to be widely used in future research fields. At the same time, new strategies and technologies should be proposed and developed to promote the application of coelenterate-type bioluminescence. [1]

C25H25N3O2

399.4849

399.195

123437-32-1

135439140

Typically exists as solid at room temperature

1.3 g/cm3

568.2ºC at 760 mmHg

297.4ºC

1.63E-13mmHg at 25°C

1.685

4.718

2

4

5

30

541

0

C1=CC=C(C=C1)CC2=NC3=C(CC4CCCC4)NC(=CN3C2=O)C5=CC=C(C=C5)O

UCSBOFLEOACXIR-UHFFFAOYSA-N

InChI=1S/C25H25N3O2/c29-20-12-10-19(11-13-20)23-16-28-24(21(26-23)14-17-8-4-5-9-17)27-22(25(28)30)15-18-6-2-1-3-7-18/h1-3,6-7,10-13,16-17,29-30H,4-5,8-9,14-15H2

2-benzyl-8-(cyclopentylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3-ol

2-benzyl-8-(cyclopentylmethyl)-6-(4-hydroxyphenyl)-7H-imidazo[1,2-a]pyrazin-3-one; CLZN-hcp; 2-benzyl-8-(cyclopentylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3-ol; Coelenterazine hcp, solid; SCHEMBL14117493; DTXSID40376337;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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