Furimazine (PBI 3939)
目录号 : GC32913Furimazine是一种咪唑并吡嗪酮类的发光底物。在哺乳动物细胞中,NanoLuc(Nluc)与Furimazine联用,产生更亮的发光,比带Coelenterazine的Oluc-19高250万倍。
Cas No.:1374040-24-0
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Kinase experiment: |
Random libraries are generated by error-prone PCR (average of 2-3 mutations per clone). Library 1 (phase 1; template=Oluc-N166R) is screened (4,400 variants) with Coelenterazine. Library 2 (phase 2; template=C1A4E) is screened (4,400 variants) with 11 novel Coelenterazine analogues: 3840, 3841, 3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900. The 11 analogues represent substitutions at positions 2, 6, and 8 and are considered to be representative of the entire set of 24 compounds; 2,200 variants are screened with compounds 3896 and 3894. All hits (improved luminescence) are screened again with the remaining Coelenterazine analogues. Library 3 (phase 3; template=C1A4E+Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the context of a mouse Id-X-HaloTag (where X=library) using Coelenterazine and Furimazine. Library screens are performed on a Freedom robotic workstation as follows: induced bacterial cultures (in 96-well microtiter plates) are lysed with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X Passive Lysis Buffer, 0.3 mg/mL lysozyme, and 0.002 units of RQ1 DNase. Assay reagent containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol, and 20 μM substrate is then added to equal volumes of lysate. Samples are measured on a GENios Pro luminometer[1]. |
References: [1]. Hall MP, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11):1848-57. |
Furimazine is an imidazopyrazinone substrate. NanoLuc (Nluc) paired with Furimazine produced 2.5 million-fold brighter luminescence in mammalian cells relative to Oluc-19 with Coelenterazine.
Furimazine is a Coelenterazine analogue.The apparent Km for purified NanoLuc (Nluc) using either Furimazine or Coelenterazine is ~10 μM, while the maximum luminescence (i.e., apparent Vmax) is ~30-fold higher for Furimazine than for native Coelenterazine. Measurement of luminescence intensity from cells expressing Nluc reveals that maximal signal is attained at about 10-20 μM Furimazine[1].
[1]. Hall MP, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012 Nov 16;7(11):1848-57.
Cas No. | 1374040-24-0 | SDF | |
Canonical SMILES | O=C1C(CC2=CC=CO2)=NC3=C(CC4=CC=CC=C4)NC(C5=CC=CC=C5)=CN31 | ||
分子式 | C24H19N3O2 | 分子量 | 381.43 |
溶解度 | DMSO : ≥ 100 mg/mL (262.17 mM) | 储存条件 | Store at 2-8°C |
General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效。 储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。 为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。 |
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Shipping Condition | 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。 |
制备储备液 | |||
1 mg | 5 mg | 10 mg | |
1 mM | 2.6217 mL | 13.1086 mL | 26.2171 mL |
5 mM | 0.5243 mL | 2.6217 mL | 5.2434 mL |
10 mM | 0.2622 mL | 1.3109 mL | 2.6217 mL |
第一步:请输入基本实验信息(考虑到实验过程中的损耗,建议多配一只动物的药量) | ||||||||||
给药剂量 | mg/kg | 动物平均体重 | g | 每只动物给药体积 | ul | 动物数量 | 只 | |||
第二步:请输入动物体内配方组成(配方适用于不溶于水的药物;不同批次药物配方比例不同,请联系GLPBIO为您提供正确的澄清溶液配方) | ||||||||||
% DMSO % % Tween 80 % saline | ||||||||||
计算重置 |
计算结果:
工作液浓度: mg/ml;
DMSO母液配制方法: mg 药物溶于 μL DMSO溶液(母液浓度 mg/mL,
体内配方配制方法:取 μL DMSO母液,加入 μL PEG300,混匀澄清后加入μL Tween 80,混匀澄清后加入 μL saline,混匀澄清。
1. 首先保证母液是澄清的;
2.
一定要按照顺序依次将溶剂加入,进行下一步操作之前必须保证上一步操作得到的是澄清的溶液,可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Novel Furimazine derivatives for nanoluciferase bioluminescence with various C-6 and C-8 substituents
Org Biomol Chem 2021 Sep 22;19(36):7930-7936.PMID:34549229DOI:10.1039/d1ob01098k.
Nanoluciferase (NLuc) is the emerging commercially available luciferase considering its small size and superior bioluminescence performance. Nevertheless, this bioluminescence system has some limitations, including narrow emission wavelength and single substrate. Herein, a series of novel Furimazine derivatives at the C-6 and C-8 positions of the imidazopyrazinone core have been designed and synthesized for extension of the bioluminescence substrates. It should be noted that two compounds, molecules A2 (2-(furan-2-ylmethyl)-6-(4-(hydroxymethyl)phenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one) and A3 (2-(furan-2-ylmethyl)-6-(4-amino-3-fluorophenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one), display reasonable bioluminescence properties for in vitro and in vivo biological evaluations. In particular, compound A3 can broaden the application of NLuc bioluminescence techniques, especially for in vivo bioluminescent imaging.
A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level
Bioconjug Chem 2022 Mar 16;33(3):496-504.PMID:35184558DOI:10.1021/acs.bioconjchem.2c00035.
Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged Furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.
Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals
Nat Methods 2020 Aug;17(8):852-860.PMID:32661427DOI:10.1038/s41592-020-0889-6.
Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate Furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
Novel NanoLuc-type substrates with various C-6 substitutions
Bioorg Med Chem Lett 2020 May 1;30(9):127085.PMID:32171617DOI:10.1016/j.bmcl.2020.127085.
NanoLuc (NLuc)-furimazine bioluminescence system offers several advantages over established systems, including improved stability, smaller size, and >150-fold enhancement in bioluminescence. Herein, we designed and synthesized a series of bioluminescent substrates with varying at the C-6 position of Furimazine for NLuc-furimazine bioluminescence system. Among all derivatives, compounds A6 and A11 provided excellent bioluminescence characteristics compared with Furimazine in vitro and in vivo. We believe that these new NLuc substrates can broaden the application of NLuc bioluminescence techniques, especially in vivo bioluminescent imaging.
Nanoluciferase signal brightness using Furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy
Cytometry A 2016 Aug;89(8):742-6.PMID:27144967DOI:10.1002/cyto.a.22870.
Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.