Visualization of long-term Mg2+ dynamics in apoptotic cells using a novel targetable fluorescent probe

Long-term Mg2+ imaging during apoptosis using a HaloTag-coupled Mg2+ probe demonstrated a Mg2+ concentration increase caused by dissociation of Mg2+ from ATP.


Materials and instruments
All chemicals used for organic synthesis were of the best grade available, supplied by Industries. 4-bromo-A23187 was purchased from Sigma-Aldrich.
GPC purifications were performed with a JAIGEL 1H-2H column (Japan Analytical Industry Co., Ltd.) using a GPC system that was comprised of a pump (LC-6AD, Shimadzu) and a detector (SPD-20A, Shimadzu). HPLC analyses were performed with an Inertsil ODS-3 (4.6 mm×250 mm) column (GL Sciences Inc.) by using an HPLC system that was comprised of a pump (PU-2080, Jasco) and a detector (MD-2010 or FP-2020, Jasco).
NMR spectra were recorded on a JEOL JNM-AL400 instrument at 400 MHz for 1 H and at 100 MHz for 13 C NMR or a Bruker Avance 500 instrument at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR, using tetramethylsilane as an internal standard. Mass spectra were measured either on a Waters LCT-Premier XE or on a JMS-700 (JEOL) mass spectrometer.
Fluorescence spectra were measured by using a Hitachi F7000 spectrometer. The slit widths were 2.5 nm for both excitation and emission, and the photomultiplier voltage was 700 V. UV-visible absorption spectra were measured using a Jasco aV-650 spectrophotometer.
For photostability analysis of MGH and Halo-TMR, light irradiation was performed using a Xe light source (MAX-303; Asahi Spectra) equipped with band pass filters (490/5 nm for MGH; 550/5 nm for Halo-TMR).

Fluorometric analysis
The relative fluorescence quantum yields of the compounds were obtained by comparing the area under the emission spectrum. The following equation was used to calculate the quantum yield: where st is the reported quantum yield of the standard, I is the integrated emission spectrum, A is the absorbance at the excitation wavelength, and n is the refractive index of the solvent.
The subscripts x and st denote the sample and the standard, respectively. Fluorescein ( = 0.85 when excited at 492 nm in 100 mM NaOH aq.) was used as the standard.

Determination of dissociation constants
The apparent dissociation constants (Kd) of MGH, HaloTag-MGH, and Magnesium Green for Mg 2+ and Ca 2+ in 100 mM HEPES buffer (pH 7.4) including 115 mM KCl and 20 mM NaCl at 37 °C were calculated using the following equation, where F is the fluorescence intensity at each metal ion concentration, Fmin is the fluorescence intensity before addition of the metal ions, and Fmax is the fluorescence intensity at the saturation state.

Preparation of HaloTag protein
Hexahistidine-tagged HaloTag was overexpressed in Escherichia coli cells, BL21 (DE3), then the cells were cultivated in Luria-Bertani medium at 37 °C. When the OD600 of the culture medium reached 0.6-0.8, the culture flask was incubated at 20 °C and isopropyl--D-thiogalactopyranoside (final concentration: 100 μM) was added to the medium. After protein expression was induced overnight, the cells were collected by centrifugation at 4,700 ×g for 12 min, and were resuspended in 50 mM sodium phosphate buffer (pH 8.0) with 300 mM NaCl. After cell lysis by sonication, the lysate was centrifuged at 32,000 ×g for 20 min. The supernatant was loaded on cOmplete His-Tag Purification Resin. After the resin was washed with 50 mM sodium phosphate buffer (pH 8.0) with 300 mM NaCl and 5 mM imidazole, proteins adsorbed on the resin were eluted using 50 mM sodium phosphate buffer (pH 8.0) with 300 mM NaCl and 250 mM imidazole. Further purification was conducted by size exclusion chromatography (Superdex TM 75 10/300 GL, GE healthcare) using 100 mM HEPES buffer (pH 7.4) with 1 mM DTT to prevent dimerization of HaloTag. The purified protein was analyzed by SDS-PAGE for the purity check.

Detection of protein labeling by SDS-PAGE
HaloTag (40 µM) was added to a solution of MGH (30 µM) in 100 mM HEPES buffer (pH 7.4) with 115 mM KCl and 20 mM NaCl at 37 °C. After incubation for 1 h, the labeled protein was denatured in 2×SDS gel loading buffer (100 mM Tris-HCl buffer (pH 6.8), 4% SDS, 20% glycerol, and 10% mercaptoethanol) and resolved by SDS-PAGE. The fluorescence image of the gel was captured using a fluorescence image analyzer (Typhoon FLA 9500, GE Healthcare Bio-Sciences AB). The gels were stained with Coomassie Brilliant Blue prior to the capture of images.
Chemiluminescence was detected using the Amersham ECL Plus Western Blotting Detection System (GE Healthcare).

Cell culture
HEK293T cells, HEK293 cells, and HeLa cells were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) plus Gluta Max-I supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere of with 5% CO2. A subculture was performed every 2-3 days from subconfluent (<80%) cultures using a trypsin-ethylenediamine tetraacetic acid solution. Transfection of plasmids was carried out in a glass-bottomed dish using Lipofectamine 3000 or 2000 according to the standard protocol.

Synthesis of compounds
Compounds 1-3 were prepared according to the previously described procedures. S3

Synthesis of 2
MeOH (120 mL) and H2SO4 (8.60 mL, 342 mmol) were added to the crude residue obtained in the preparation of compound 1, and the mixture was stirred for 3 days at reflux temperature. After cooling, the salts were filtered off and the solvent was evaporated. The residue was dissolved in ethyl acetate and washed with 2 M NaOH aq. and then with brine. After the organic layer was dried over Na2SO4 and evaporated under reduced pressure, the residue was purified by flash column chromatography on silica gel (ethyl acetate/hexane = 3:7).

Synthesis of 3
Compound 2 (837 mg, 2.57 mmol) was dissolved in AcOH (10 mL), and fuming HNO3 (160 µL, 3.86 mmol) dissolved in AcOH (1.0 mL) was added in a dropwise manner at 0 °C for over 5 min. After confirming the completion of the reaction, the reaction mixture was poured into ice-water. After extraction with DCM, the organic layer dried over with Na2SO4 and filtered. After removing of the solvent under reduced pressure, the residue was purified by column chromatography on silica gel (DCM/MeOH = 99.5:0.5). Compound 3 (738 mg, 77%) was obtained as a yellow solid. 1

Synthesis of 4
Compound 3 (700 mg) was dissolved in 20 mL of MeOH/H2O (3:1) and 3 mL of 2 M NaOH aqueous solution was added dropwise at 0 °C. The reaction mixture was then warmed to room temperature. After stirring for 6 h, Dowex-50 H + resin was added into the reaction mixture and the pH was adjusted to 5-6, then was filtered off, and the solvent was removed under reduced pressure. Compound 4 (620 mg) was obtained as an orange powder.

Synthesis of 5
Compound 4 (620 mg, 1.88 mmol) was dissolved in dry DMF (10.0 mL). Bromomethyl acetate (1.41 mL, 15.0 mmol) and dry TEA (3.15 mL, 22.6mmol) were added at room temperature under Ar. After stirring for 1 day, the solvent was removed under reduced pressure, and DCM was added to the residue, and washed with water. The organic layer was washed with brine, dried with Na2SO4 and evaporated. The residue was purified by GPC.
Compounds 7-9 were prepared according to the previously described procedures. S4 Synthesis of 7 4-Chlororesorcinol (4.89 g, 33.8 mmol) and 4-carboxyphthalic anhydride (3.25 g, 16.9 mmol) were stirred in methanesulfonic acid (40 mL) at 90 °C for 14 h. The reaction mixture was then poured into 400 mL of stirred ice water, and the resulting suspension was filtered The residue was washed with H2O and dried under vacuum at 90 °C overnight to give a brown solid (7.58 g). The product was carried forward without further purification.

Synthesis of 8
The brown solid (7.58 g) was stirred in 25 mL of acetic anhydride and 1.5 mL of pyridine and heated to reflux for 30 min. The reaction mixture was cooled to room temperature for 4 h and then filtered. The filtrate was added slowly into 75 mL of stirred H2O, and the mixture was stirred for additional 10 min, and then extracted with ethyl acetate. The combined organic layer was washed with 0.4 M HCl aq. and brine, dried with Na2SO4, and evaporated to give compound 8 (4.40 g, 49%) as a yellow solid. 1

Synthesis of 9
Compound 8 (2.20 g, 4.16 mmol) was dissolved in 24 mL of MeOH/H2O (3:1). 2 M NaOH aq. (4.5 mL) was added dropwise at 0 °C. The color of the solution changed quickly from yellow to orange. The reaction mixture was warmed at room temperature. After stirring for 2 h, Dowex-50 H + resin was added into the reaction mixture and the pH was adjusted to 5-6, then was filtered off, and the solvent was removed under reduced pressure. Compound 9 (1.85 g, quant.) was obtained as an orange powder. 1
Compound 9 (1.50 g, 3.37 mmol) dissolved in 28 mL acetonitrile and 28 mL H2O was added to the reaction solution, and the mixture was refluxed for 3 h. The reaction mixture was cooled, and the solvent was removed under reduced pressure to give compound 10 (1.30 g) as a red solid. The product was carried forward without further purification.

Synthesis of 12
Chromium trioxide (25.0 g, 162 mmol) was dissolved in 300 mL of 1.5 M H2SO4 aq., and the solution was cooled to 0 °C. 2-[2-(chloroethoxy)ethoxy]ethanol (8.30 g, 49.2 mmol) in 150 mL of acetone was dropwisely added, and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, and the aqueous layer was extracted with DCM. The combined organic layer was washed with brine and dried with Na2SO4 and evaporated. Compound 12 (4.89 g, 42%) was obtained as a colorless oil. 1

Synthesis of 13
Compound 12 (3.38 g, 18.5 mmol) and NaN3 (4.81 g, 74.0 mmol) in 13 mL of H2O were stirred with heating at 80 °C for 32 h. After cooling to room temperature, the reaction mixture was acidified with 2 M HCl aq. and extracted with DCM. The combined organic layer was dried with Na2SO4 and evaporated. Compound 13 (2.39 g, 68%) was obtained as a colorless