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

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(µ-O)Ru(NH₃)₄(OH₂)]Cl₅

1. Synthesis of [Ru(NH₃)₅Cl]Cl₂ ¹²
   1. Dissolve 1.00 g of RuCl₃·nH₂O (40% Ru by weight, 4.1 mmol) in 5 mL of H₂O. Cool the dark brown solution to 0 °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.
   2. 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.
   3. Heat this solution at 105 °C while stirring for 1-2 h. A yellow solid will precipitate out of solution. When no more precipitate visibly forms, remove from heat.
   4. Allow the reaction mixture to cool to room temperature, and then place in a 0 °C ice bath for 10 min. Collect the yellow solid by vacuum filtration and wash with 5 mL each of ethanol and diethyl ether.
   5. 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.
   6. 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.
2. Synthesis of [(OH₂)(NH₃)₄Ru(µ-O)Ru(NH₃)₄(OH₂)]Cl₅
   1. 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 °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.
3. Purification by cation-exchange chromatography
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. Use glass beads to cover the top of the resin. These will prevent the resin from being disturbed when new solutions are added.
   6. Elute the column with 20 mL of 1 M HCl.
   7. Elute the column with an increased HCl concentration of 1.5 M (≈ 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.
   8. 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.
4. Characterization and verification of purity of [(OH₂)(NH₃)₄Ru(µ-O)Ru(NH₃)₄(OH₂)]Cl₅
   1. Test all of the fractions from step 1.3.8. by UV-vis spectroscopy. To accomplish this task, add 100 µL 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.
   2. 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.
   3. 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.
   4. 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.
5. 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.
   ​NOTE: [(OH₂)(NH₃)₄Ru(µ-O)Ru(NH₃)₄(OH₂)]Cl₅ will be referred to as [Ru] in this section
   1. 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 µM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-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.
   2. Culture HeLa cells in 500 cm² Petri dishes in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO₂ at 37 °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.
      1. 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
   3. 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 × g. Decant the supernatant and add the calculated volume of BGSS. Resuspend cells gently.
      1. 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.
         ​NOTE: Calcium Green-5N is light-sensitive. Store in the dark and minimize light exposure.
   4. Setup the fluorimeter to excite at 506 nm and read the emission at 532 nm with the cuvette-holder controlled at 37 °C. Prepare an acrylic cuvette with a stir bar or wheel, 1.8 mL cell suspension from 1.5.2 above, 1.8 µL digitonin solution, 3.6 µL Calcium Green-5N (solution), and 9 µL [Ru] (for 1 mM stock solution, 5 µM final concentration). Incubate cells for 15 min in the fluorimeter.
      1. 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.
      2. 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.
      3. 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 µL of CaCl₂ (10 µM 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 (≈5 min). Add additional calcium boluses to determine the mitochondrial calcium uptake response of cells not treated with [Ru].
   5. In another cuvette containing 5 µM [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.