A universal fluorogenic switch for Fe(ii) ion based on N-oxide chemistry permits the visualization of intracellular redox equilibrium shift towards labile iron in hypoxic tumor cells

Oxygen-dependent fluctuation of labile Fe(ii) was visualized by a new N-oxide-based fluorescent probe for Fe(ii) ion.

Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2017 S2 General: All chemicals used in this study were commercial products of the highest available purity and were further purified by the standard methods, if necessary. 1 H-NMR spectra were obtained on a JEOL ECA-500 spectrometer at 500 MHz and JEOL JNM AL-400 spectrometer at 400 MHz. 13 C-NMR spectra were obtained on a JEOL ECA-500 spectrometer at 125 MHz and JEOL AL-400 spectrometer at 100 MHz. Chemical shifts of 1 H-NMR are referenced to tetramethylsilane (TMS). Chemical shifts of 13 C-NMR are referenced to CDCl 3 (77.0) or CD 3 OD (49.0). Chemical shifts and coupling constants were recorded in units of ppm and Hz, respectively. ESI-mass spectra were measured on a JEOL JMS-T100TD mass spectrometer. High-resolution mass spectra (HRMS) were measured on a JEOL JMS-T100TD by using polyethyleneglycol (PEG) as an internal standard. Reactions were monitored by silica gel TLC (Merck Silica gel 60 PF 254 ) with visualization of components by UV light (254 nm) or with visual observation of the dye spots. Products were purified on a silica gel column chromatography (Taiko-shoji AP-300S).

Steady-state absorption and fluorescence spectroscopy
The UV-vis absorption spectra were recorded on an Agilent 8453 photodiode array spectrometer equipped with a UNISOKU thermo-static cell holder (USP-203). Fluorescence spectra were recorded using a JASCO FP6600 with a slit width of 5 nm and 6 nm for excitation and emission, respectively. To reduce fluctuation in the excitation intensity during measurement, the lamp was kept on for 30 min prior to the experiment. The path length was 1 cm with a cell volume of 3.0 mL. Quantum yields were measured in 50 mM HEPES buffer (pH 7.4) by using a Quantaurus-QY absolute photo-luminescence quantum yields measurement system (C11347-01, Hamamatsu Photonics).
For determination of pK a , absorption spectra were measured in 200 mM sodium phosphate buffer adjusted to various pH values. pH profiles at 450 nm (FluNox-1 and FluNox-2) and 575 nm (SiRhoNox-1) were fitted to the equation as follows: where pK a is the acid dissociation constants of FluNox derivatives: A 0 is the initial absorbance at 450 nm at pH 2.03, A 1 is the maximum absorbance at 450 nm associated with the corresponding pK a values. The pK a value for SiRhoNox-1 could not be calculated because of no pH dependent change in absorbance spectrum of SiRhoNox-1 (see Figure S3).

Product analysis
To a solution of a probe (100 µM) in 50 mM HEPES buffer (pH 7.4) was added a solution of FeSO 4 (final, 1 mM). The mixture was kept for 1 h under an ambient condition. The products were analyzed with LC-MS system (LC-20AD, Shimadzu) equipped with a photodiode-array detector (SPD-M20A, Shimadzu) and an LCMS-IT-TOF mass spectrometer (Shimadzu) and with Waters X-Bridge C 18 column (3.5 µm, 2.1 × 100 mm) eluted with gradient systems consisting of H 2 O/CH 3 CN as described in the caption of Figure S5. The retention times were compared with those of the parent dyes in 50 mM HEPES buffer (pH 7.4). Assignments of the compounds were based on the observed m/z values at each peak.

Confocal fluorescence imaging experiments
Confocal fluorescence images were acquired with Zeiss LSM700 laser confocal microscopy or Olympus IX83 microscope equipped with a 130 W mercury lump, an EMCCD camera (Hamamatsu Photonics, ImagEM), and a disk scan confocal unit (DSU). Images were obtained with appropriate filter sets for each dye as follows.

Imaging experiments under hypoxia
Hypoxia treatments were performed in a hypoxia chamber (InvivO2 hypoxia workstation, Baker Ruskinn, USA) under control of oxygen concentration (1% or 5%). All the mediums and solvents used for hypoxic treatments were pre-incubated for more than 8 h prior to use in the chamber, and all the treatments with SiRhoNox-1, Bpy, and DPI were performed in the hypoxia chamber. HepG2 cells (8 × 10 4 ) were seeded on each well of Advanced-TC glass bottom dishes. After incubation in MEM (+10% FBS, 2 mM glutamine, and 1% ABAM) for 2 days prior to use, the cells were placed in the hypoxia chamber and incubated for 2, 4, 8, or 12 h under the controlled oxygen concentration of 1% or 5% at 37 °C. For the cell-staining experiments, the cells were washed with HBSS, and then the medium was replaced with new HBSS solution containing 5 µM SiRhoNox-1with or without 1 mM Bpy or DPI (10 or 100 µM, diphenyliodonium chloride). After incubation of the cells in the chamber for 30 min, the cells were washed with HBSS and then imaged. For control experiments (20% O 2 ), the cells were incubated in an incubator (37 °C, 5% CO 2 , 95% air) for the same period, and all the treatments were performed in a standard clean bench.

Quantification of total intracellular iron by atomic absorption spectrometry
HepG2 cells (1.2 × 10 5 cells) were seeded on a 10 cm dish 2 days prior to use. The cells were cultured under hypoxia (1% O 2 ) as described above for 0, 2, 4, 8, and 12 h. Then, the medium was washed with cold phosphate-buffered saline (PBS, 3 mL × 3). The cells were removed from the dishes by using a scraper, and then the suspension was centrifuged (1,000 rpm, 5 min). The supernatant was carefully removed, and the cells were re-suspended into conc. HNO 3 (100 µL). The suspension was heated to 90 °C for 4 h to dissolve the cell bodies. The lysate was diluted to 2 mL with distilled water. Concentrations of iron in the samples were measured by furnace atomic absorption spectroscopy with a Shimadzu AA-7000 atomic absorption spectrometer. The obtained values (ng/mL) were normalized with the cell numbers (per 10 6 cells).
Total 4 dishes were prepared for each experiment as described above. 3 dishes were used for iron quantification, and the rest was used to calculate the number of cells. To determine the cell numbers, the cells were collected by trypsinization at the same time point with the other 3 dishes, and the number of the cells was counted by a hematocytometer.

Preparation and imaging study of spheroids 6
HepG2 cells (1.0 × 10 3 cells/well) were seeded on a PrimeSurface ® 96-well plate (Sumitomo Bakelite Co. Ltd., Japan). After incubation for 5 days in MEM (+10% FBS), the spheroids growing to 500 µm of diameter were picked up and transferred to a new PrimeSruface ® 96-well plate. Then the spheroids were treated with 10 µM SiRhoNox-1 or 200 µM pimonidazole hydrochloride (Hypoxyprobe, Inc., USA) in MEM (−FBS, 0.5% DMSO) at 37 °C for 2 h. The medium was exchanged to fresh medium (MEM, −FBS), and the spheroids were S10 incubated for further 30 min. Then, the spheroids were fixed by treatment with a mixture of formalin (10%) and sucrose (10%) in water at room temperature for 2 h. After washing with PBS, the spheroids were embedded into optimal cutting temperature (O.C.T.) compound (Sakura Finetek Japan) and frozen at −20 °C overnight.
The embedded spheroids were cut into 7 µm thick slices with a cryostat (Leica). The sections placed on slide glasses without holes in the central area were selected for next step.