钌线粒体钙吸收抑制剂的合成与评价
Summary
提出了一种用于线粒体钙吸收的钌抑制剂的合成、纯化和表征的协议。本文介绍了一种评价其在化哺乳动物细胞中的有效性的方法。
Abstract
我们详细介绍了线粒体钙吸收抑制剂的合成和纯化, [(oh2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(OH2)5 +。这种化合物的优化合成开始从 [Ru (NH3)5cl] cl2在 1 M NH4OH 在一个封闭的容器, 产生一个绿色的解决方案。纯化是用阳离子交换色谱法完成的。这种化合物的特点和验证是纯紫外-可见光和红外光谱。荧光光谱法对化 HeLa 细胞线粒体钙摄取抑制特性进行了评价。
Introduction
线粒体钙是对正常细胞功能, 包括能量生产和细胞凋亡至关重要的一些过程的关键调节器。1,2,3线粒体钙 uniporter (MCU), 一种离子转运蛋白, 驻留在线粒体膜上, 调节钙离子流入线粒体。4,5,6单片机的化学抑制剂是继续努力研究这种转运蛋白和线粒体钙的功能和细胞作用的宝贵工具。化合物 [(小贩2) (nh3)4ru (µ) 汝 (nh3)4(o2CH)3 +, Ru360, 是唯一已知的单片机的选择性抑制剂之一, 其报告的 Kd值为24µM.7 ,8,9,10此复合物是在钌红色 (RuRed) 的商业配方中发现的常见杂质, triruthenium µ-羰基桥接 hexacation 的公式 [(NH3)5ru (µ) 汝 (nh3)4(µ) ru (nh3)5)6 +, 这也被用作钙吸收抑制剂。虽然 Ru360 是商用的, 但成本很高。此外, Ru360 的合成和分离受到困难的纯化程序和模糊的表征方法的挑战。
我们最近报告的替代程序访问一个 Ru360 模拟, [(OH2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(oh2)] Cl5。11此复合抑制具有高亲和力的 MCU, 类似于 Ru360。在本协议中, 我们将描述这一化合物的最有效的合成, 它从 [Ru (NH3)5cl] cl2开始。使用强酸性阳离子交换树脂提纯产品, 并对此程序的常见缺陷进行了详细介绍。我们还提出了表征和评估化合物纯度的方法, 并描述了一个简单的方法来测试它的有效性, 阻断线粒体钙摄取。
Protocol
Representative Results
Discussion
线粒体钙吸收抑制剂 [(OH2) (nh3)4汝 (µ O) ru (nh3)4(OH2)] cl5可以从 [Ru (NH3)5cl] cl2中合成众所周知的钌 (III)起始材料, 如本程序所述。合成的 [Ru (NH3)5cl] cl2很容易实现, 但难度很小。在水合肼中搅拌 RuCl3 16 小时后, 溶液的 pH 值应调整为2与 HCl 的数值。pH 值下降是实现所需产品的关键。如果?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了康奈尔大学的支持。这项工作利用康奈尔材料研究中心的共享设施, 这是支持通过 NSF MRSEC 计划 (赠款 DMR-1120296)。S.R.N. 承认, 支持由 NSF 研究生研究奖学金 (赠款 DGE-1650441) 和 Dr. 戴夫 Holowka 协助钙实验。在这一材料中发表的任何意见、发现、结论或建议都是作者的观点, 不一定反映国家科学基金会的观点。
Materials
| Ruthenium Trichloride hydrate | Pressure Chemical | 3750 | |
| Concentrated hydrochloric acid | J.T. Baker | 9535 | |
| Concentrated ammonium hydroxide | Mallinckrodt Chemical Works | A669C-2 1 | |
| Dowex 50 WX2 200-400 Mesh | Alfa Aesar | 13945 | |
| Calcium Green 5N | Invitrogen | C3737 | |
| Digitonin | Aldrich | 260746 | |
| DMSO | Aldrich | 471267 | |
| EGTA | Aldrich | E3889 | |
| KCl | USB | 20598 | |
| KH2PO4 | Aldrich | P3786 | |
| MgCl2 | Fisher Scientific | M33-500 | |
| HEPES | Fluka | 54466 | |
| Sodium Succinate | Alfa Aesar | 33386 | |
| EDTA | J.T. Baker | 8993-01 | |
| Glucose | Aldrich | G5000 | |
| 200 Round bottom flask | ChemGlass | CG-1506-14 | |
| Glass stopper | ChemGlass | CG-3000-05 | |
| 10 mm x 15 cm glass column with reservoirs | Custom – similar to Chemglass columns | Similar to CG-1203-20 | |
| DMEM | Corning | 10-017-CV | |
| FBS | Gibco | 10437028 | |
| PBS | Corning | 21-040-CV | |
| Round bottom Falcon tubes | Fisher Scientific | 14-959-11B | |
| 500 cm2 petri dishes | Corning | 431110 | |
| Trypan blue | ThermoFisher Scientific | 15250061 | |
| Hemacytometer | Aldrich | Z359629 | |
| Acrylic Cuvettes | VWR | 58017-875 | |
| UV-Vis spectrometer | Agilent Model Cary 8454 | ||
| Spectrofluorimeter | SLM Model 8100C | ||
| IR spectrometer | Bruker Hyprion FTIR with ATR attachment | ||
| Centrifuge | ALC Model PM140R | ||
| Inverted light microscope | VWR | 89404-462 |
Riferimenti
- De Stefani, D., Rizzuto, R., Pozzan, T. Enjoy the trip: Calcium in mitochondria back and forth. Annu. Rev. Biochem. 85, 161-192 (2016).
- Contreras, L., Drago, I., Zampese, E., Pozzan, T. Mitochondria: the calcium connection. Biochim. Biophys. Acta. 1797 (6-7), 607-618 (2010).
- Giorgi, C., et al. Mitochondrial calcium homeostasis as potential target for mitochondrial medicine. Mitochondrion. 12 (1), 77-85 (2012).
- De Stefani, D., Raffaello, A., Teardo, E., Szabò, I., Rizzuto, R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature. 476 (7360), 336-340 (2011).
- Baughman, J. M., et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature. 476 (7360), 341-356 (2011).
- Kamer, K. J., Mootha, V. K. The molecular era of the mitochondrial calcium uniporter. Nat. Rev. Mol. Cell Biol. 16 (9), 545-553 (2015).
- Ying, W. -. L., Emerson, J., Clarke, M. J., Sanadi, D. R. Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex. Biochimica. 30 (20), 4949-4952 (1991).
- Emerson, J., Clarke, M. J., Ying, W. -. L., Sanadi, D. R. The component of “ruthenium red” responsible for inhibition of mitochondrial calcium ion transport. Spectra, electrochemistry, and aquation kinetics. Crystal structure of µ-O-[(HCO2)(NH3)4Ru]2Cl3. J. Am. Chem. Soc. 115 (25), 11799-11805 (1993).
- Matlib, M. A., et al. Oxygen-bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitro and in Situ in Single Cardiac Myocytes. J. Biol. Chem. 273 (17), 10223-10231 (1998).
- Oxenoid, K., et al. Architecture of the mitochondrial calcium uniporter. Nature. 533 (7602), 269-273 (2016).
- Nathan, S. R., et al. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake. Inorg Chem. 56 (6), 3123-3126 (2017).
- Allen, A. D., Senoff, C. V. Preparation and infrared spectra of some ammine complexes of ruthenium(II) and ruthenium(III). Can. J. Chem. 45 (12), 1337-1341 (1967).
- Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., Fiskum, G. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. USA. 93 (18), 9893-9898 (1996).
- Deak, A. T., et al. Assessment of mitochondrial Ca⁺ uptake. Meth. Molec. Biol. 1264, 421-439 (2015).
