Luminescent enzyme substrate

用扩展的电子偶联取代C-8位的亚甲基是一种很有前途的制备红移酮肠嗪衍生物的新方法。在本文中，我们描述了一种含氧的coelenterazine衍生物，相对于coelenterazine 400a具有显著的红移(63 nm)生物发光信号最大值(DeepBlueC™，1)。在细胞成像中，含硫coelenterazine衍生物显示出显著的(1.77±0.09;P≤0.01)，其发光信号略高于coelenterazine 400a，含氧coelenterazine衍生物的发光信号略高于(0.74±0.08;P≤0.05)较低的发光信号。这有助于进一步了解生物发光的潜在机制。[3]

相对量子产率(RQY)与离体发光动力学[3]
RQY研究和动力学分析使用IVIS动力学进行，该动力学由安装在避光标本室(暗箱)上的冷却电荷耦合器件(CCD)摄像机、摄像机控制器、摄像机冷却系统和计算机控制组成。数据表示为光强(蓝色最弱，红色最强)叠加在灰度参考图像上的伪彩色图像(以光子/s/cm2/scr为单位)。在区域上绘制圆形指定感兴趣区域(roi)，并使用Living Image软件将光输出量化为每秒发射的光子总数。 为确定底物的适宜不饱和量，采用10 μL coelenterazine (CTZ) 衍生物1 - 3 (1 ~ 100 μmol/L)和90 μL DMSO或Rluc酶，终浓度为15 nmol/L(生物发光)。将10 μL coelenterazine (CTZ) 衍生物(终浓度为5 μmol/L)与90 μL DMSO或Rluc酶(15 nmol/L)混合在96孔黑板上，以防止孔间光反射，测定RQY和反应动力学。在混合后立即测量发光信号，并使用IVIS监测25-30分钟(发光几乎衰减到接近背景水平)。化学发光每5分钟记录一次光输出，曝光时间30 s;生物发光每1分钟记录一次光输出，前15分钟曝光时间5 s。采用Prism 5.0 GraphPad软件对采集数据进行分析，计算总光输出。在相同条件下，用Tris-HCl缓冲液代替coelenterazine (CTZ) 衍生物溶液作为相应的空白对照。所有试验均为三份。

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活细胞发光活性测定[3]
表达Rluc的ES-2细胞(人卵巢癌细胞系)由BioDiagnosis提供。ES-2细胞在Dulbecco's modified Eagle's培养基(DMEM;含10%胎牛血清(FBS)和0.5 μg/mL嘌呤霉素的高葡萄糖(含l-谷氨酰胺)，在37℃的湿化气氛中，在5% CO2培养箱中培养。coelenterazine (CTZ) 衍生物在乙醇中溶解成1 mmol/L的原液，用Tris-HCl缓冲液稀释至梯度浓度(5、10、20、40、60、80、100 μmol/L)。 ES-2-Rluc细胞生长于黑色96孔板(每孔4 × 104个细胞)。24小时孵育后，取出培养基。然后用Tris-HCl缓冲液冲洗细胞2次，并用100 μL浓度的coelenterazine (CTZ) 衍生物溶液(范围为0 ~ 100 μmol/L)处理细胞。然后使用IVIS立即确定生物发光信号。每1分钟记录一次光输出，曝光时间30秒，直到发光几乎衰减到接近背景水平。测量每个孔的发光信号(每秒光子数)并绘制平均值。所有的实验都是三次重复。

[1]. Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J Am Chem Soc. 2007 Oct 3;129(39):11900-1.

[2]. The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J Recept Signal Transduct Res. Feb-Nov 2002;22(1-4):533-41.

[3]. Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation. Chinese Chemical Letters, 2016, 27(4): 550-554.

In conclusion, we have described an oxygen-containing CTZ derivative 2 and it exhibited a more significant red-shifted (63 nm) bioluminescence signal maximum relative to coelenterazine 400a, while it had lower quantum yield. The results of this study appear to support our hypothesis that the introduction of oxygen heteroatom at the C-8 position could also produce a bathochromic effect. While a possible mechanism of the CTZ luminescence reaction has been proposed (Fig. S2 in Supporting information), the enzymatic recognition mechanism of the Rluc system is not yet fully understood. We may surmise that it is due to an efficient extension of p-electron conjugation, electronegativity, or H-bonds of oxygen at the C-8 position. We also developed a new measurement method through an IVIS Kinetic equipped with a CCD to examine RQY. The RQY of CTZ derivative 3 for chemiluminescence is in good agreement with previously reported value. To get a genuine and correct RQY in bioluminescence, we employed commercially available purified Rluc enzyme in place of the rough cytosolic extract to avoid enzyme-independent luminescence. Compounds 2 and 3 showed lower quantum yield that is quite different from previously reported value. However, we discovered compound 3 had a higher affinity to Rluc than coelenterazine 400a. In cell imaging, compound 3 also displayed a higher luminescence signal. In a word, compounds 2 and 3 are promising bright red-shifted CTZ derivatives that provide a novel approach to improve the luminescence properties of CTZ analogs. More significantly, it is beneficial to understand the underlying mechanisms of Rluc bioluminescence upon reacting with coelenterazine.[3]

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In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.[2]

C26H22CLN3O

427.925384998322

427.145

2320429-05-6

Coelenteramine 400a;70217-82-2

137700438

Typically exists as solid at room temperature

50.4

2

3

5

31

524

0

UVZGQDPDAXEWQO-UHFFFAOYSA-N

InChI=1S/C26H21N3O.ClH/c30-26-23(17-20-12-6-2-7-13-20)28-25-22(16-19-10-4-1-5-11-19)27-24(18-29(25)26)21-14-8-3-9-15-21;/h1-15,18,30H,16-17H2;1H

2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3-ol;hydrochloride

6-phenyl-2,8-bis(phenylmethyl)-imidazo[1,2-a]pyrazin-3(7H)-one,monohydrochloride; 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3-ol;hydrochloride; Coelenteramine 400a (hydrochloride); EX-A9184A; Coelenteramine 400a hydrochloride;

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。