A mitochondria-targeting fluorescent probe for detection of mitochondrial labile Fe(II) ion

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Electronic Supplementary Material (ESI) for Metallomics. This journal is The Royal Society of Chemistry 2018 A mitochondria-targeting fluorescent probe for detection of mitochondrial labile Fe(II) ion Tasuku Hirayama*, Satoki Kadota, Masato Niwa, Hideko Nagasawa Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University Contents 1. Steady state absorption and fluorescence measurements... S2 2. Product analysis... S3 3. Supporting figures Figure S1 UV-vis absorption spectra of MtFluNox and MtRhodol... S4 Figure S2 HPLC-mass analysis of the reaction between MtFluNox and Fe(II)... S4 Figure S3 Fluorescence response of MtFluNox over various biological molecules... S5 Figure S4 Fluorescence microscopic images of co-staining experiments... S5 Figure S5 Fluorescence imaging of succinylacetone-induced accumulation of Fe(II)... S5 4. 1 H-and 13 C-NMR spectra of the newly synthesized compounds... S7

1. Steady state absorption and fluorescence measurements The UV-vis absorption spectra were recorded on an Agilent 8453 photodiode array UV-vis spectrometer. Fluorescence spectra were recorded using a JASCO FP6600 with a slit width of 5 nm and 6 nm for excitation and emission, respectively. The path length was 1 cm with a cell volume of 3.0 ml. Excitation was provided at 510 nm. For all the fluorescence measurements, MtFluNox was used at a final concentration of 2 µm (from 1 mm stock solution in DMSO) in 50 mm HEPES buffer (ph 7.4). 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). Fluorescence responses of MtFluNox to various metal ions were measured as follows. An aqueous solution of transition metal ion species (stock solutions: 10 mm for MnSO 4, CoSO 4, NiSO 4, FeSO 4, FeCl 3, CuSO 4, and ZnSO 4 ; 1 M for NaCl, KCl, MgCl 2, and CaCl 2 ), or [Cu(CH 3 CN) 4 ]PF 4 (from 10 mm stock solution in MeCN) was added to give the final concentrations of 1 mm for Na(I), K(I), Mg(II), and Ca(II) and 20 µm for other metal ion species. In the case of Cu(I), MtFluNox was reacted with Cu(I) in the presence of 200 µm glutathione. The mixtures were kept at room temperature for 1 h and then applied to fluorescence measurement. Selectivity for reductants, reactive oxygen species, transferrin, and effect of chelator were tested under the conditions as follows. Na 2 S 2 O 3 : 100 µm from 100 mm stock solution in water Sodium ascorbate : 1 mm from 100 mm stock solution in water Cysteine : 1 mm from 100 mm stock solution in water Glutathione : 1 mm from 100 mm stock solution in HEPES buffer (ph was adjusted to 7.4) NaNO 2 : 100 µm from 100 mm stock solution in water O 2 : 100 µm from saturated KO 2 solution in DMSO (ca. 1 mm) 1) H 2 O 2 : 100 µm from 100 mm stock solution in water OH : 200 µm H 2 O 2 and 20 µm FeSO 4 (1 mm stock solution of MtFluNox in DMF was used instead of DMSO) NaOCl : 100 µm from 100 mm stock solution in water NO : 100 µm NOC-5 from 10 mm stock solution in 0.1 M NaOH aq. hemin : 20 µm hemin from 5 mm stock solution in DMSO. Fe(II)+Bpy : 20 µm FeSO 4 in the presence of 200 µm 2,2'-bipyridyl (Bpy) form 100 mm stock solution in DMSO. Fe(II)+DFO : 20 µm FeSO 4 FeSO 4 in the presence of 200 µm deferroxamine (DFO) form 100 mm stock solution in water. MtFluNox (2 µm) was incubated under each condition in 50 mm HEPES buffer (ph 7.4, 0.2% DMF as co-solvent) for 1 h, and then fluorescence spectra were measured, respectively. S2

2. Product analysis To a solution of a probe (100 µm) in 50 mm HEPES buffer (ph 7.4, 5%DMSO as co-solvent) 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 (Chromaster 5110, Hitachi High-tech) equipped with a photodiode-array detector (Chromaster 5430, Hitachi High-tech) and a mass spectrometer (Chromaster 5610 MS Detector, Hitachi High-tech) and with Waters symmetry C 18 column (3.5 µm, 4.6 75 mm) eluted with a gradient system consisting of H 2 O (solvent A) and CH 3 CN (solvent B) containing 0.05% formic acid; 20%B to 70%B over 20 min. The retention times were compared with those of the parent dyes in 50 mm HEPES buffer (ph 7.4, 5%DMSO as co-solvent). Assignments of the compounds were based on the observed m/z values at each peak. Reference 1) A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078 10080. S3

3. Supporting figures Figure S1 (a) UV-vis absorption spectra of MtFluNox (2 µm, black line) and MtRhodol (2 µm, red line). (b) UV-vis absorption change of MtFluNox (2 µm) upon the addition of Fe(II) (20 µm; as FeSO 4 ). Each spectrum was recorded every 5 min. All the spectra were obtained in HEPES buffer (50 mm, ph 7.4, 0.2%DMSO as co-solvent). Figure S2 HPLC-mass monitoring of the reaction between MtFluNox (100 µm) and Fe(II) ion (1 mm). (a) MtFluNox, (b) the reaction mixture of MtFluNox and Fe(II), and (c) MtRhodol (as the parent fluorophore). The detailed LC condition is described in the experimental section 3. Product analysis. S4

Figure S3 Fluorescence response of MtFluNox against various reductants, reactive oxygen species. Bars represent relative fluorescence intensities at 535 nm. Figure S4 (a) Colocalization assay of MtFluNox and MitoTracker Deep Red FM in HepG2 cells in the absence or presence of Fe(II). (b) Colocalization assay of MtRhodol and MitoTracker Deep Red FM in HepG2 cells in the absence or presence of Fe(II). Pearson's correlation values (Rcoloc) for each experiment are shown (n = 5). Scale bars indicate 25 µm. Figure S5 Fluorescence microscopic images of mitochondrial labile Fe(II) in HEK293 cells treated with (a) vehicle, (b) succinylacetone (SA; 1 mm), or (c) SA in the presence of DFO (100 µm). The cells were stained with Ac-MtFluNox (1 µm, 1 h). (d f) DIC images of the same fields of view of (a c), S5

respectively. Scale bars indicate 25 µm. (g) Quantification of the images taken under each condition (n = 10). Statistical analysis was performed by Student's t-test. **P < 0.05 (n = 6). Error bars indicate ±S.E.M. S6

4. 1 H- and 13 C-NMR spectra of the newly synthesized compounds 3-O-Methoxymethyl-6-(4-tert-butoxycarbonylpyperazin-1-yl)rhodol (Boc-piperazinylrhodol, 2) 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 0.99 0.99 1.01 3.02 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 8.024 8.009 7.659 7.610 7.264 7.170 7.155 6.954 6.950 6.700 6.696 6.692 6.642 6.609 5.200 3.582 3.573 3.563 3.481 3.209 1.485 0.000 1.00 1.02 2.03 4.01 3.07 3.00 4.12 8.99 0 1.0 2.0 3.0 4.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 169.207 158.495 154.365 152.801 152.458 152.200 152.143 134.735 129.423 128.831 128.516 126.580 124.644 123.690 112.463 112.292 111.929 109.173 103.307 102.009 94.007 83.066 79.737 77.248 77.200 77.000 76.742 55.901 47.870 28.164 S7

Piperazinylrhodol-triphenylphosphonium bromide salt (MtRhodol, 4) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 7.830 7.827 7.822 7.814 7.806 7.790 7.710 7.702 7.695 7.686 7.099 7.083 6.682 6.666 6.661 6.544 6.526 6.489 6.477 6.473 4.912 3.613 3.526 3.426 3.306 3.302 3.300 3.297 3.177 3.148 1.06 17.05 1.00 6.04 6.06 4.01 1.97 2.00 2.650 1.897 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 171.825 171.215 161.657 154.198 154.103 153.941 153.283 136.228 136.209 134.797 134.721 131.535 131.430 131.096 120.022 119.336 113.307 111.524 110.694 103.664 102.815 79.475 49.334 49.162 49.000 48.828 48.657 45.805 42.352 33.548 33.405 22.560 22.150 19.298 S8

MtFluNox PF 6 salt 0 1.0 2.0 3.0 0.97 1.00 0.94 0.94 0.93 1.00 1.02 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 1.07 4.00 1.05 1.94 1.95 2.07 2.00 8.098 8.014 7.890 7.875 7.854 7.844 7.814 7.767 7.744 7.735 7.719 7.206 7.191 6.973 6.737 6.732 6.631 6.614 6.584 6.580 6.567 4.867 4.649 4.622 4.106 4.086 4.009 3.413 3.307 3.303 3.300 3.254 2.786 2.710 1.958 1.946 16.09 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 0.1 0.2 0.3 140.0 130.0 120.0 110.0 136.924 136.304 136.285 134.864 134.788 131.592 131.535 131.488 130.620 130.171 127.501 126.089 125.145 122.159 120.175 119.488 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 116.713 114.280 111.257 110.551 171.959 170.976 161.447 156.516 154.132 153.473 153.044 136.304 136.285 134.864 134.788 131.592 131.535 131.488 130.620 130.171 126.089 125.145 120.175 119.488 103.655 83.596 79.465 68.000 67.705 49.172 49.000 48.828 48.657 41.331 37.764 33.271 33.138 22.550 22.131 19.269 S9

Ac-MtRhodol (5) 0 1.0 2.0 3.0 4.0 1.04 0 1.0 2.0 3.0 4.0 8.022 8.007 1.04 8.0 7.9 7.8 7.7 7.6 7.889 7.874 7.864 7.849 17.15 7.790 7.778 7.775 7.762 7.697 7.688 7.681 7.672 7.632 7.616 7.602 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 7.889 7.874 7.864 7.849 7.778 7.703 7.697 7.688 7.681 7.672 17.15 7.273 7.054 7.050 6.777 6.773 6.768 6.694 6.689 6.654 6.635 6.605 1.04 4.034 4.024 3.795 3.740 3.342 3.332 3.255 3.244 3.121 2.312 17.15 2.030 2.017 1.999 1.986 1.922 0.000 1.07 2.05 1.07 1.02 1.03 1.05 0 1.0 2.0 3.0 4.0 2.05 1.02 1.03 1.05 7.2 7.1 7.0 6.9 6.8 6.7 6.6 7.185 7.170 7.054 7.050 1.07 6.794 6.777 6.773 6.768 6.755 2.05 2.04 1.97 1.98 6.694 6.689 1.02 6.654 6.635 1.03 6.605 6.601 6.588 6.584 2.01 2.03 2.01 1.05 3.00 2.01 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 134.907 134.688 134.669 133.410 133.333 130.0 120.0 130.195 130.090 129.575 128.679 128.431 126.237 124.663 123.795 118.368 117.681 116.966 116.546 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 170.323 169.121 168.654 152.563 152.238 151.895 151.742 151.552 134.688 134.669 133.410 133.333 130.195 130.090 128.431 118.368 117.681 111.929 110.022 108.639 101.809 82.513 77.258 77.210 77.000 76.742 47.841 47.555 44.579 40.859 32.303 32.160 21.516 21.105 20.867 18.063 S10

Ac-MtFluNox 0.96 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 0 0.1 0.2 17.94 8.2 8.1 8.0 8.127 8.122 0.96 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 7.848 7.832 7.822 7.807 7.736 7.730 7.723 7.720 7.714 7.706 7.699 7.213 7.205 7.201 7.034 7.016 6.914 6.897 6.892 6.874 6.857 4.891 4.644 4.617 8.042 8.027 4.103 4.078 4.013 3.644 3.608 3.411 3.303 3.251 2.785 2.706 2.274 0.96 1.973 1.949 1.912 17.94 0 0.1 0.2 0.3 0.4 17.94 1.99 7.2 7.1 7.0 6.9 6.8 7.213 7.205 7.201 0.93 1.99 7.034 7.016 0.93 6.914 6.910 6.897 6.892 6.874 6.857 1.99 1.05 4.02 1.03 0.93 2.06 2.00 1.04 1.04 3.00 2.09 0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 137.115 136.285 136.266 134.855 134.778 131.755 131.573 131.478 130.600 130.0 120.0 130.019 126.947 126.289 125.087 121.787 120.166 119.755 119.479 117.466 117.266 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 111.743 111.390 171.930 170.661 170.509 156.850 154.084 154.017 152.605 136.285 136.266 134.855 134.778 131.573 131.478 130.019 126.289 120.166 119.479 111.743 111.390 82.403 68.058 68.000 67.800 67.743 49.172 49.000 48.828 48.657 41.331 37.764 33.271 33.128 22.512 22.092 20.891 19.259 S11

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