Name: Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor (Video) | JoVE Journal
Uploaded: 2017-10-26T15:00:00.000+00:00
Duration: 7 min 12 s
Description: Learn to synthesize and purify a ruthenium-based mitochondrial calcium uptake inhibitor that is ten times more effective than commercial alternatives. This method enables potent research tools for studying mitochondrial biology.

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5 minutes

This research was supported by Cornell University. This work made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (Grant DMR-1120296). S.R.N. acknowledges support by an NSF Graduate Research Fellowship (Grant DGE- 1650441) and Dr. Dave Holowka for assistance with the calcium experiments. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

NOTE: Concentrated acids and bases are used in this synthesis. Use all appropriate safety practices when performing the reaction including the use of engineering controls (fume hood) and personal protective equipment (PPE) including safety glasses, gloves, lab coat, full length pants, and closed-toe shoes.
1. Preparation of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
<ol>
	<li>Synthesis of [Ru(NH3)5Cl]Cl2 12
	<ol>
		<li>Dissolve 1.00 g of RuCl3&#183;nH2O (40% Ru by weight, 4.1 mmol) in 5 mL of H2O. Cool the dark brown solution to 0 &#176;C in an ice bath. Add 11 mL (0.23 mol) of 80% hydrazine hydrate solution in a dropwise manner. The initial reaction will be vigorous with the evolution gas, resulting in a brown solution. Let the resulting solution stir at room temperature for 16 h; the final solution will be dark red. 
		Caution: Hydrazine is acutely toxic and carcinogenic. Additionally, anhydrous forms of this reagent are explosive. As always, use appropriate PPE and fume hoods when handling. Do not concentrate these solutions to dryness.</li>
		<li>To this solution, add approximately 5-10 mL of concentrated HCl to adjust the pH to 2. At this point, the solution will be yellow-brown in color.</li>
		<li>Heat this solution at 105 &#176;C while stirring for 1-2 h. A yellow solid will precipitate out of solution. When no more precipitate visibly forms, remove from heat.</li>
		<li>Allow the reaction mixture to cool to room temperature, and then place in a 0 &#176;C ice bath for 10 min. Collect the yellow solid by vacuum filtration and wash with 5 mL each of ethanol and diethyl ether.</li>
		<li>Completely dissolve the crude product in 15-25 mL of hot water. Chill 10 mL of a concentrated HCl solution in a filter flask by placing it in an ice bath. Filter the yellow solution into the chilled HCl solution to induce precipitation of a pale yellow pure solid. Filter this precipitate and wash with 5 mL each of 0.5 M HCl, ethanol and ether.</li>
		<li>Characterize the compound using IR spectroscopy. Verify purity by the identification of stretching frequencies at 3226 cm-1, 1604 cm-1, 1297 cm-1, and 801 cm-1. A common minor impurity at 2069 cm-1 is assigned to [Ru(NH3)5N2]Cl3.</li>
	</ol>
	</li>
	<li>Synthesis of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
	<ol>
		<li>Dissolve 100 mg (0.34 mmol) [Ru(NH3)5Cl]Cl2 in 50 mL of 1 M NH4OH in a 200 mL heavy wall round-bottomed pressure vessel. Loosely cap the flask with a stopper and heat the reaction mixture at 75 &#176;C for 6 h. Remove from heat and stir at room temperature for 4 days to yield a dark green solution. 
		Caution! Heating a sealed vessel results in a pressure build-up. Make sure to use appropriate pressure-safe glassware. For this reaction, the purpose of sealing the vessel is to minimize loss of gaseous NH3. Therefore, place the stopper loosely to allow for release of excess pressure.</li>
	</ol>
	</li>
	<li>Purification by cation-exchange chromatography
	<ol>
		<li>In a 25 mL beaker, suspend 5 g cation-exchange resin (e.g., Dowex 50WX2 200-400 mesh (H+ form) in 10 mL 0.1 M HCl.</li>
		<li>Load this slurry into a 10 mL column (10 mm diameter, 15 cm height) affixed with a 50 mL solvent reservoir. Wash the resin with approximately 20-30 mL of 0.1 M HCl, until the eluate is colorless.</li>
		<li>Return to the green reaction solution isolated in step 1.2.1. To this solution, add concentrated HCl to adjust the pH to 2, at which point the solution color changes to brown.</li>
		<li>Load this acidified solution to the cation-exchange resin column prepared in step 1.3.2 by gently pipetting it on top of the resin. Let the eluate completely drain, and continue loading the solution. Repeat this process until the entire solution has been added. The top of the resin will be dark brown/black. The resin will decrease in volume slightly.</li>
		<li>Use glass beads to cover the top of the resin. These will prevent the resin from being disturbed when new solutions are added.</li>
		<li>Elute the column with 20 mL of 1 M HCl.</li>
		<li>Elute the column with an increased HCl concentration of 1.5 M (&#8776; 50 mL). A yellow solution will begin to come off the column. Increase the HCl concentration to 2 M and continue eluting until the eluate is colorless or a very pale green-yellow. A total volume of 150-200 mL will be required for this process.</li>
		<li>Increase the HCl concentration to 2.5 M (20-50 mL). Collect the eluate as fractions in test tubes. Increase to 3 M HCl. The product will elute from the column as a green-brown solution. A red-brown fraction may also begin to come off of the column. As these fractions are oxidized ruthenium red impurities, do not pool with the green-brown fractions.</li>
	</ol>
	</li>
	<li>Characterization and verification of purity of [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5
	<ol>
		<li>Test all of the fractions from step 1.3.8. by UV-vis spectroscopy. To accomplish this task, add 100 &#181;L of a given fraction into 2 mL of 3 M NH3 and analyze by UV-vis spectroscopy. Fractions containing pure product will have a large absorbance band at 360 nm and a less intense absorbance at 600 nm. Absorbance at 480 or 533 nm is indicative of oxidized ruthenium red and ruthenium red impurities, respectively.</li>
		<li>Pool fractions containing pure product and evaporate the solution to dryness by rotary evaporation. The product will be isolated as a green-brown solid. Yields are typically on the order of 5-15 mg (10-20% yield). Single-crystals, suitable for X-ray diffraction, can be obtained by the vapor diffusion of ethanol into aqueous solutions of the compound.</li>
		<li>To verify purity, analyze the compound by UV-vis spectroscopy in a solution of pH 7.4 phosphate-buffered saline (PBS). Purity may be assessed by taking the ratio of intensity of the 360 nm and 600 nm peaks. This ratio is 31 for a pure compound. For impure compounds, the ratio will be smaller.</li>
		<li>Analyze the sample in the solid-state by IR spectroscopy. Diagnostic bands are at 3234 cm-1, 3151 cm-1, 1618 cm-1, 1313 cm-1, and 815 cm-1. Common impurity bands are seen at 1762 cm-1 and 1400 cm-1, characteristic of NH4Cl. Ruthenium red can be identified by bands at 1404 cm-1, 1300 cm-1, 1037 cm-1 and 800 cm-1.</li>
	</ol>
	</li>
	<li>Evaluation of mitochondrial calcium uptake inhibition by fluorescence spectroscopy 
	Caution! The following procedures use mammalian cells. Work should be carried out in appropriate laminar flow hoods that are certified for biological safety level 2 (BSL2) research. 
	&#8203;NOTE: [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 will be referred to as [Ru] in this section
	<ol>
		<li>Make buffered glucose-containing saline solution (BGSS) as the assay media. BGSS is a solution comprising 110 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM sodium succinate, 30 &#181;M ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA). Combine everything except the EGTA, adjust pH to 7.4. Add EGTA and readjust pH to 7.4. For 50 mL of assay media add 0.5 mL of 1 mg/mL glucose.</li>
		<li>Culture HeLa cells in 500 cm2 Petri dishes in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO2 at 37 &#176;C. Amplify HeLa cells growing in a 100 mm Petri dish by seeding them in a 500 cm2 petri dish. The total media volume in the large dish is 115 mL. Each large dish will yield approximately 18 million cells, enough for two fluorescence spectroscopy experiments.
		<ol>
			<li>Grow the cells until they reach 90-95% confluency. Remove media, and rinse the cells with 15 mL pH 7.4 PBS. Add 15 mL of 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS and incubate for 10 min to detach the cells. Transfer the cells to 14 mL round bottom falcon tubes</li>
		</ol>
		</li>
		<li>Count cells using trypan blue and a hemocytometer with an inverted microscope, and calculate the total number of cells and the volume of media needed to reach 7.5 million cells per 1.8 mL volume of medium. Centrifuge the cells for 10 min at 5310 &#215; g. Decant the supernatant and add the calculated volume of BGSS. Resuspend cells gently.
		<ol>
			<li>For this assay, prepare stock solutions of 40 mM digitonin in dimethyl sulfoxide (DMSO), 1 mM Calcium Green-5N in H2O, and 10 mM CaCl2 in H2O. [Ru] stock solutions, prepared in pure water, can range from 1-3 mM. 
			&#8203;NOTE: Calcium Green-5N is light-sensitive. Store in the dark and minimize light exposure.</li>
		</ol>
		</li>
		<li>Setup the fluorimeter to excite at 506 nm and read the emission at 532 nm with the cuvette-holder controlled at 37 &#176;C. Prepare an acrylic cuvette with a stir bar or wheel, 1.8 mL cell suspension from 1.5.2 above, 1.8 &#181;L digitonin solution, 3.6 &#181;L Calcium Green-5N (solution), and 9 &#181;L [Ru] (for 1 mM stock solution, 5 &#181;M final concentration). Incubate cells for 15 min in the fluorimeter.
		<ol>
			<li>Read the data as the excitation/emission ratio instead of the raw absorption. This practice minimizes errors associated with fluctuations in the light source intensity.</li>
			<li>Carry out the first sample analysis in the absence of [Ru] to measure the effect of the addition of CaCl2 on the response of the cells.</li>
			<li>Begin analysis on the fluorimeter with the settings described in 1.5.4. Wait approximately 2 minutes to establish a stable emission baseline, and then add 1.8 &#181;L of CaCl2 (10 &#181;M final concentration). The emission intensity will increase immediately upon the addition of the CaCl2, and will then decay over the course of minutes as the calcium ions enter the mitochondria. Wait until the decay has finished (&#8776;5 min). Add additional calcium boluses to determine the mitochondrial calcium uptake response of cells not treated with [Ru].</li>
		</ol>
		</li>
		<li>In another cuvette containing 5 &#181;M [Ru], repeat the experiment as described above in 1.5.4.3. In the presence of the inhibitor, emission intensity will increase, but not decay. This observation signifies blocked mitochondrial calcium uptake.</li>
	</ol>
	</li>
</ol>

<ol><li>De Stefani, D., Rizzuto, R., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enjoy+the+trip%3A+Calcium+in+mitochondria+back+and+forth%5BTitle%5D)+AND+%22Annu.+Rev.+Biochem%22%5BJournal%5D)">Enjoy the trip: Calcium in mitochondria back and forth.</a> Annu. Rev. Biochem. 85, 161-192 (2016).</li>
<li>Contreras, L., Drago, I., Zampese, E., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria%3A+the+calcium+connection%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">Mitochondria: the calcium connection.</a> Biochim. Biophys. Acta. 1797 (6-7), 607-618 (2010).</li>
<li>Giorgi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+calcium+homeostasis+as+potential+target+for+mitochondrial+medicine%5BTitle%5D)+AND+%22Mitochondrion%22%5BJournal%5D)">Mitochondrial calcium homeostasis as potential target for mitochondrial medicine.</a> Mitochondrion. 12 (1), 77-85 (2012).</li>
<li>De Stefani, D., Raffaello, A., Teardo, E., Szabò, I., Rizzuto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+forty-kilodalton+protein+of+the+inner+membrane+is+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter.</a> Nature. 476 (7360), 336-340 (2011).</li>
<li>Baughman, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+genomics+identifies+MCU+as+an+essential+component+of+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.</a> Nature. 476 (7360), 341-356 (2011).</li>
<li>Kamer, K. J., Mootha, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+era+of+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">The molecular era of the mitochondrial calcium uniporter.</a> Nat. Rev. Mol. Cell Biol. 16 (9), 545-553 (2015).</li>
<li>Ying, W. -L., Emerson, J., Clarke, M. J., Sanadi, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+mitochondrial+calcium+ion+transport+by+an+oxo-bridged+dinuclear+ruthenium+ammine+complex%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Inhibition of mitochondrial calcium ion transport by an oxo-bridged dinuclear ruthenium ammine complex.</a> Biochemistry. 30 (20), 4949-4952 (1991).</li>
<li>Emerson, J., Clarke, M. J., Ying, W. -L., Sanadi, D. R. <a target="_blank" href="https://www.researchgate.net/publication/244437951_The_component_of_ruthenium_red_responsible_for_inhibition_of_mitochondrial_calcium_ion_transport_Spectra_electrochemistry_and_aquation_kinetics_Crystal_structure_of_m-O-HCO2NH34Ru2Cl_3">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.</a> J. Am. Chem. Soc. 115 (25), 11799-11805 (1993).</li>
<li>Matlib, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen-bridged+Dinuclear+Ruthenium+Amine+Complex+Specifically+Inhibits+Ca2%2B+Uptake+into+Mitochondria+in+Vitro+and+in+Situ+in+Single+Cardiac+Myocytes%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Oxygen-bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitro and in Situ in Single Cardiac Myocytes.</a> J. Biol. Chem. 273 (17), 10223-10231 (1998).</li>
<li>Oxenoid, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Architecture+of+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Architecture of the mitochondrial calcium uniporter.</a> Nature. 533 (7602), 269-273 (2016).</li>
<li>Nathan, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+Methods+for+the+Preparation+of+a+Functional+Analogue+of+Ru360%2C+a+Potent+Inhibitor+of+Mitochondrial+Calcium+Uptake%5BTitle%5D)+AND+%22Inorg+Chem%22%5BJournal%5D)">Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake.</a> Inorg Chem. 56 (6), 3123-3126 (2017).</li>
<li>Allen, A. D., Senoff, C. V. <a target="_blank" href="http://www.nrcresearchpress.com/doi/abs/10.1139/v67-220#.WW5AYYjyvIU">Preparation and infrared spectra of some ammine complexes of ruthenium(II) and ruthenium(III).</a> Can. J. Chem. 45 (12), 1337-1341 (1967).</li>
<li>Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., Fiskum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bcl-2+potentiates+the+maximal+calcium+uptake+capacity+of+neural+cell+mitochondria%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.</a> Proc. Natl. Acad. Sci. USA. 93 (18), 9893-9898 (1996).</li>
<li>Deak, A. T., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25631032">Assessment of mitochondrial Ca⁺ uptake.</a> Meth. Molec. Biol. 1264, 421-439 (2015).</li>
</ol>

The authors have nothing to disclose

The mitochondrial calcium uptake inhibitor [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5 can be synthesized from [Ru(NH3)5Cl]Cl2, a well known ruthenium(III) starting material, as described in this procedure. The synthesis of [Ru(NH3)5Cl]Cl2 is readily achieved with little difficulty. After stirring RuCl3 for 16 h in hydrazine hydrate, the pH of the solution should be adjus...

Mitochondrial calcium is a key regulator for a number of processes that are critical to normal cell function, including energy production and apoptosis.1,2,3 The mitochondrial calcium uniporter (MCU), an ion transporter protein that resides on the inner mitochondrial membrane, regulates the influx of calcium ions into the mitochondria.4,5,6 Chemical inhibitors of the MCU are valuable tools for continuing efforts to study the function and cellular roles of this transport protein and mitochondrial calcium. The compound [(HCO2)(NH3)4Ru(&#181;-O)Ru(NH3)4(O2CH)]3+, Ru360, is one of the only known selective inhibitors for the MCU with a reported Kd value of 24 &#181;M.7,8,9,10 This complex is a common impurity found in commercial formulations of ruthenium red (RuRed), a triruthenium di-&#181;-oxo bridged hexacation of the formula [(NH3)5Ru(&#181;-O)Ru(NH3)4(&#181;-O)Ru(NH3)5)]6+, which has also been used as a calcium uptake inhibitor. Although Ru360 is commercially available, it is very costly. Moreover, the synthesis and isolation of Ru360 is challenged by difficult purification procedures and ambiguous characterization methods.
We have recently reported alternative procedures to access a Ru360 analogue, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]Cl5.11 This compound inhibits the MCU with high affinity, similar to Ru360. In this protocol, we will describe our most effective synthesis of this compound, which commences from [Ru(NH3)5Cl]Cl2. Purification of the product using strongly acidic cation-exchange resin is detailed, along with common pitfalls for this procedure. We also present methods for characterization and assessment of compound purity, and delineate a simple approach to test its efficacy in blocking mitochondrial calcium uptake.

<table><tbody><tr><td>Ruthenium Trichloride hydrate</td><td>Pressure Chemical</td><td>3750</td><td></td></tr><tr><td>Concentrated hydrochloric acid</td><td>J.T. Baker</td><td>9535</td><td></td></tr><tr><td>Concentrated ammonium hydroxide</td><td>Mallinckrodt Chemical Works</td><td>A669C-2 1</td><td></td></tr><tr><td>Dowex 50 WX2 200-400 Mesh</td><td>Alfa Aesar</td><td>13945</td><td></td></tr><tr><td>Calcium Green 5N</td><td>Invitrogen</td><td>C3737</td><td></td></tr><tr><td>Digitonin</td><td>Aldrich</td><td>260746</td><td></td></tr><tr><td>DMSO</td><td>Aldrich</td><td>471267</td><td></td></tr><tr><td>EGTA</td><td>Aldrich</td><td>E3889</td><td></td></tr><tr><td>KCl</td><td>USB</td><td>20598</td><td></td></tr><tr><td>KH2PO4</td><td>Aldrich</td><td>P3786</td><td></td></tr><tr><td>MgCl2</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>HEPES</td><td>Fluka</td><td>54466</td><td></td></tr><tr><td>Sodium Succinate</td><td>Alfa Aesar</td><td>33386</td><td></td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td></td></tr><tr><td>Glucose</td><td>Aldrich</td><td>G5000</td><td></td></tr><tr><td>200 Round bottom flask</td><td>ChemGlass</td><td>CG-1506-14</td><td></td></tr><tr><td>Glass stopper</td><td>ChemGlass</td><td>CG-3000-05</td><td></td></tr><tr><td>10 mm x 15 cm glass column with reservoirs</td><td>Custom - similar to Chemglass columns</td><td>Similar to CG-1203-20</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Round bottom Falcon tubes</td><td>Fisher Scientific</td><td>14-959-11B&amp;nbsp;</td><td></td></tr><tr><td>500 cm&lt;sup&gt;2&lt;/sup&gt; petri dishes</td><td>Corning</td><td>431110</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Hemacytometer</td><td>Aldrich</td><td>Z359629</td><td></td></tr><tr><td>Acrylic Cuvettes</td><td>VWR&amp;nbsp;</td><td>58017-875</td><td></td></tr><tr><td>UV-Vis spectrometer</td><td>Agilent Model Cary 8454&amp;nbsp;</td><td></td><td></td></tr><tr><td>Spectrofluorimeter</td><td>SLM Model 8100C</td><td></td><td></td></tr><tr><td>IR spectrometer</td><td>Bruker Hyprion FTIR with ATR attachment</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>ALC Model PM140R</td><td></td><td></td></tr><tr><td>Inverted light microscope</td><td>VWR&amp;nbsp;</td><td>89404-462</td><td></td></tr></tbody></table>

synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

This method describes a synthesis of the mitochondrial calcium uptake inhibitor [(OH2)(NH3)4Ru(µ-O)Ru(NH3)4(OH2)]Cl5 starting from [Ru(NH3)5Cl]Cl2, a well known ruthenium(III) starting material. [Ru(NH3)5Cl]Cl2 is characterized by IR spectroscopy, with vibrational modes at 3200 cm-1, 1608 cm-1, 1298 cm-1, and 798 cm...

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

synthesis-evaluation-ruthenium-based-mitochondrial-calcium-uptake

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Learn to synthesize and purify a ruthenium-based mitochondrial calcium uptake inhibitor that is ten times more effective than commercial alternatives. This method enables potent research tools for studying mitochondrial biology.

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

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