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1 Supporting Information Ratiometric Fluorescent Sensor Based on Inhibition of Resonance for Detection of Cadmium in Aqueous Solution and Living Cells Lin Xue,, Guoping Li,, Qing Liu,,, Huanhuan Wang, Chun Liu, Xunlei Ding, Shenggui He,* and Hua Jiang,* Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, , P. R. China State Key Laboratory of Fine Chemicals, Department of Chemical Engineering, Dalian University of Technology, 158 Zhongshan Road, Dalian , P. R. China Graduate School of Chinese Academy of Sciences, Beijing, , P. R. China *To whom correspondence should be addressed. Contents Theoretic Calculations...2 Determination of Protonation Constants...4 Job s Plot...5 Metal Ion Selectivity....6 ph Effect on the Fluorescence Properties of DQCd1 and Cd-DQCd1...7 Determination of Apparent Dissociation Constant (K d )...7 Calculation of Detection Limit H NMR spectra of DQCd1 and Zn-DQCd Crystallographic Data...9 Cytotoxicity Assays in NIH 3T3 Cells...10 Confocal Fluorescence Microscopy NMR spectrum S1

2 Theoretic Calculations. Density functional theory (DFT) calculations S1 were performed using the Gaussian 03 programm S2 with the B3LYP exchange-correlation functional. All-electron triple-ξ valence basis sets with polarization functions (6-311G**) are used for all atoms. Geometry optimizations were performed with full relaxation of all atoms. Calculations for compound 1 and 1 H + were performed in gas phase without solvent effects, and the protonation of compound 1 is simulated by adding one proton to the nitrogen atom in the quinoline moiety. Vibrational frequency calculations were performed to check that the stable structures had no imaginary frequency. Charge distribution was obtained by Mulliken population. Electronic transition energies and oscillator strengths were calculated with TDDFT method. Table S1. Calculated charge distributions for compound 1 and 1 H + No. atom Compound 1 1 H + Charge increase [a] 1 C C C C C C N C C C O C C N C C O C C C C Charge increase on oxygen atom of isobutoxy group Total charge increase on isobutoxy group [a] positive charge distribution increase after compound 1 protonated. Table S2. Main singlet vertical electron transition energies ( E) and wavelengths (λ), oscillator strengths (F), and configuration interaction coefficients (CI coeff ) for compound 1. Label Transition E [ev] (λ [nm]) F Excited state (CI coeff ) A S0 S (336) ( ) B S0 S (292) ( ) S2

3 C S0 S (247) ( ) D S0 S (243) ( ) ( ) E S0 S (234) ( ) F S0 S (228) ( ) ( ) G S0 S (221) ( ) Figure S1. Normalized UV-Vis and TDDFT singlet monoelectronic vertical transitions calculated for compound 1. Transitions with oscillator strength lower than are not reported for clarity. For transition labeling, see Table S2. Table S3. Main singlet vertical electron transition energies ( E) and wavelengths (λ), oscillator strengths (F), and configuration interaction coefficients (CI coeff ) for compound 1 H +. Label Transition E [ev] (λ [nm]) F Excited state (CI coeff ) A S0 S (441) ( ) B S0 S (328) ( ) ( ) C S0 S (287) ( ) D S0 S (239) ( ) E S0 S (232) ( ) F S0 S (225) ( ) ( ) S3

Figure S2. Normalized UV-Vis spectrum and TDDFT singlet monoelectronic vertical transitions calculated for compound 1 H +. Transitions with oscillator strength lower than 0.

(a) Emission spectra of 1 (10 µm) and (b) absorption spectra of 1 (20 µm) at various ph values in aqueous solution (10 mm HEPES, 0.

4 Figure S2. Normalized UV-Vis spectrum and TDDFT singlet monoelectronic vertical transitions calculated for compound 1 H +. Transitions with oscillator strength lower than are not reported for clarity. For transition labeling, see Table S3. Determination of Protonation Constants. Figure S3. (a) Emission spectra of 1 (10 µm) and (b) absorption spectra of 1 (20 µm) at various ph values in aqueous solution (10 mm HEPES, 0.1 M NaCl) Insert: emission intensity (λ em = 554 nm) and absorbance of 1 (λ abs = 400 nm) over the ph range 2 11, the solid lines represent the non-linear least-squares fits to the experimental data. Figure S4. (a) Emission spectra of DQCd1 (10 µm) and (b) absorption spectra of DQCd1 (20 µm) at various ph values in aqueous solution (10 mm HEPES, 0.1 M NaCl) Insert: emission intensity (λ em = 558 nm) and absorbance of DQCd1 (λ abs = 425 nm) over the ph range 2 11, the solid lines represent the non-linear least-squares fits to the experimental data. S4

5 Figure S5. (a) Absorbance (λ abs = 405 nm) and (b) Fluorescence intensity (λ ex = 405 nm, λ em = 558 nm) of DQCd1 as a function of DQCd1 concentration in buffer solution. Figure S6. The fluorescence spectra of DQCd1 (10 µm) in buffer solution (10 mm HEPES, 0.1 M NaCl, ph = 7.4, 2 ml) was kept at room temperature (25 ± 5 C). The fluorescence spectra (λ ex = 405 nm) was measured after 0, 5, 13, 25, 30 days. Before each measurement, the titration cell was maintained at 25 C by using a water bath for 30 min. Job s Plot S3 Figure S7. Job s plot for DQCd1 in buffer solution (10 mm HEPES, 0.1 M NaCl, ph = 7.4). (a) The sum of DQCd1 and Cd 2+ concentrations is 25 µm; (b) The sum of DQCd1 and Zn 2+ concentrations is 25 µm. S5

6 Metal Ion Selectivity. Figure S8. (a) Metal ion selectivity profiles of DQCd1 (10 µm) in the buffer solution (10 mm HEPES, 0.1 M NaCl, ph = 7.4): Gray bars represent the fluorescence intensity at 495 nm of DQCd1 in the presence of the metal ions (10 µm Mn 2+, Fe 2+, Co 2+, Ni 2+, Cu 2+, Zn 2+, Cd 2+, Hg 2+, Pb 2+, and 1.0 mm Mg 2+, Ca 2+, and K + ); Black bars represent the fluorescence intensity at 495 nm of DQCd1 in the presence of the indicated metal ions, followed by 10 µm of Cd 2+. (b) Fluorescence intensity (λ ex = 405 nm, λ em = 495 nm) of DQCd1 (10 µm) containing 10 µm Zn 2+ after addition of 10 µm Cd 2+ (10 µm) as a function of time. Figure S9. Metal ion selectivity profiles of DQCd1 (10 µm) in the presence of 50 µm of NTA: Gray bars represent the fluorescence intensity at 495 nm of DQCd1 in the presence of the metal ions (10 µm Mn 2+, Fe 2+, Co 2+, Ni 2+, Cu 2+, Zn 2+, Cd 2+, Hg 2+, Pb 2+, and 1.0 mm Mg 2+, Ca 2+, and K + ); Black bars represent the fluorescence intensity at 495 nm of DQCd1 in the presence of the indicated metal ions, followed by 10 µm of Cd 2+, λ ex = 405 nm, 10 mm HEPES, 50 µm NTA, 0.1 M NaCl, ph = 7.4. Figure S10. (a) Fluorescence spectra of DQCd1 (10 µm) in the absence and presence of 10 µm Cd 2+, 10 µm Zn 2+, 0.1mM Zn 2+ in buffer solution (10 mm HEPES, 500 µm NTA, 0.1 M NaCl, ph = 7.4). (b) Gray bars represent the ratio F 495nm /F 558nm of DQCd1 (10 µm) in the presence of the metal ions (10 µm Cd 2+, 10 µm S6

Zn 2+, 0.1mM Zn 2+ ); Black bars represent the ratio of DQCd1 in the presence of the indicated metal ions, followed by 10 µm of Cd 2+, λ ex = 405 nm, 10 mm HEPES, 500 µm NTA, 0.1 M NaCl, ph = 7.4. Figure S11.

(b) Emission intensity F 495nm of DQCd1 (10 µm) in the presence of 10 µm CdCl 2 and 10 µm various counter-anions. ph Effect on the Fluorescence Properties of DQCd1 and Cd-DQCd1. Figure S12.

7 Zn 2+, 0.1mM Zn 2+ ); Black bars represent the ratio of DQCd1 in the presence of the indicated metal ions, followed by 10 µm of Cd 2+, λ ex = 405 nm, 10 mm HEPES, 500 µm NTA, 0.1 M NaCl, ph = 7.4. Figure S11. (a) Fluorescence spectra of DQCd1 (10 µm) in the presence of 10 µm CdCl 2 and 10 µm various counter-anions (10 mm HEPES, 0.1 M NaCl, ph = 7.4). (b) Emission intensity F 495nm of DQCd1 (10 µm) in the presence of 10 µm CdCl 2 and 10 µm various counter-anions. ph Effect on the Fluorescence Properties of DQCd1 and Cd-DQCd1. Figure S12. (a) Fluorescence spectra of DQCd equiv Cd 2+ at various ph values in aqueous solution (10 mm HEPES, 0.1M NaCl). (b) Fluorescence intensity of Cd-DQCd1 (10 µm DQCd1 + 1 equiv of Cd 2+, λ em = 495 nm) at various ph values in buffer (10 mm HEPES, 0.1 M NaCl). (c) Ratio value F 495nm /F 558nm of DQCd1 and Cd-DQCd1 at various ph values. λ ex = 405 nm. Determination of Apparent Dissociation Constant (K d ). Calculation of free Cd 2+ concentrations [Cd 2+ ] free A series of buffer solution (10 mm HEPES, 0.1 M NaCl, ph = 7.4) were prepared that contained 1 mm EDTA (ethylenediaminetetraacetic acid), 5 mm Mg 2+. The logk and pka values of EDTA were taken from literatures S4 (logk (Cd-EDTA) = 16.5, logk (Mg-EDTA) = 8.79 (25 C, I = 0.1) and pk a1 = 10.19, pk a2 = 6.13, pk a3 = 2.69, pk a4 = 2.0). Protonation constants must be corrected upward by 0.11 when working at 0.1 M ionic strength. Free Cd 2+ concentrations were obtained using the program Hyss S5. S7

8 [Cd 2+ ] total (mm) [Cd 2+ ] free (nm) [Cd 2+ ] total (mm) [Cd 2+ ] free (nm) Calculation of free Zn 2+ concentrations [Zn 2+ ] free A series of buffer solution (10 mm HEPES, 0.1 M NaCl, ph = 7.4) were prepared that contained 2 mm NTA (Nitrilotriacetic acid). The logk and pk a values of NTA were taken from the literature S6 (logk (Zn-NTA) = (25 C, I = 0.1) and pk a1 = 9.65, pk a2 = 2.48, pk a3 = 1.8). Protonation constants must be corrected upward by 0.11 when working at 0.1 M ionic strength. Free Zn 2+ concentration was calculated using the method described in ref. S7. [Zn 2+ ] total (mm) [Zn 2+ ] free (nm) [Zn 2+ ] total (mm) [Zn 2+ ] free (nm) Calculation of Detection Limit. The detection limit was determined from the fluorescence titration data based on the reported method. S8 According to the result of titrating experiment, the ratio fluorescence responses (F 495nm /F 558nm ) were normalized between the minimum and the maximum value. A linear regression curve was then fitted to these ratio fluorescence responses data, and the point at which this line crossed the ordinate axis was considered as the detection limit ( M). Figure S13. The limit of detection (LOD) of DQCd1: ratio fluorescence responses (F 495nm /F 558nm ) as a function of Cd 2+ concentration, The solid line represent a linear fit to the experimental data. 1 H NMR spectra of DQCd1 and Zn-DQCd1. S8

9 Figure S14. Partial 1 H NMR spectra (400 MHz) of (a) DQCd1 (5 mm) and (b) DQCd1 + Zn 2+ (5 mm) in DMSO-d 6 Figure S15. Fluorescence spectra (λ ex = 405 nm) of 10 µm DQCd1 upon the titration of 10 µm Cd 2+ ( ) and 10 µm Zn 2+ ( ) in aqueous acetonitrile buffer (10 mm HEPES, 0.1 M NaCl, 50%CH 3 CN, ph = 7.4). Crystallographic Data. Table S4. Crystallographic parameters for complex Cd-DQCd1 and Zn-DQCd1. S9

10 Compound Cd-DQCd1 Zn-DQCd1 Formula C 62 H 80 N 10 O 5 Cd 3 Cl 6 C 29 H 39 N 7 O 10 Zn Formula weight Wavelength(Å) Crystal system Triclinic Triclinic Space group P 1 P 1 T(K) 113(2) 113(2) a(å) (3) (17) b(å) (3) (3) c(å) (4) (5) α( ) 92.27(3) 83.71(3) β( ) (3) 89.65(3) γ( ) (3) 78.83(3) V(Å 3 ) (12) (11) Z 2 4 D(Mg/m 3 ) F(000) µ(mo Ka)(mm 1 ) θ range(º) 1.40 ~ ~ Goodness of fit on F R 1, wr 2 [I > 2σ (I)] , , R indices (all data) , , CCDC. No R = F 0 Fc / F 0, wr 2 = { [w(f 2 0 F 2 c ) 2 ]/ [w(f 2 0 ) 2 ]} 1/2 Cytotoxicity Assays in NIH 3T3 Cells. To ascertain the cytotoxic effect of sensor treatment over a 24-h period, NIH 3T3 cells were passed and plated to ~60% confluence in 12-well plates 16 h before treatment and bathed in 1 ml of DMEM. Prior to DQCd1 treatment, the DMEM was removed and replaced with fresh DMEM, and aliquots of DQCd1 stock solutions (5 mm DMSO) were added for final concentrations of 5, 10, and 20 µm. The treated cells were incubated for 24 h at 37 C and under 5% CO 2. Subsequently, the cells were treated with Cell Counting Kit-8 (CCK-8, Dojin Laboratories Kumamoto, Japan) (100 µl/well) and incubated for an additional 2 3 h (37 C, 5% CO 2 ). The absorbance at 450 nm was recorded using a microplate reader. Three independent trials were conducted, and the averages and standard deviations are reported. The reported percent cell survival values are relative to untreated control cells. Additional control studies indicated that the DMSO added (1 4 µl) as a result of sensor treatment has no effect on cell viability under these conditions. S10

7±3 87.9±3 a Cell viability was quantified by the CCK-8 assay (mean ± SD). Confocal Fluorescence Microscopy.

Confocal fluorescence and bright-field images of NIH 3T3 cells stained with DQCd1 (5 µm) and Lysotracker Red (1 µm)

(a) Bright-field transmission image; (b) Fluorescence image of DQCd1 observed with excitation at 405 nm; (c)

Confocal fluorescence images of intracellular Cd 2+ in HEK293 cells with DQCd1.

11 Table S5. Cytotoxicity Data of DQCd1 (NIH 3T3 cells, 24h) a Sensor Concentration 5 µm 10 µm 20 µm % Cell Survival 100.8±4 98.7±3 87.9±3 a Cell viability was quantified by the CCK-8 assay (mean ± SD). Confocal Fluorescence Microscopy. Figure S16. Confocal fluorescence and bright-field images of NIH 3T3 cells stained with DQCd1 (5 µm) and Lysotracker Red (1 µm) for 30 min. (a) Bright-field transmission image; (b) Fluorescence image of DQCd1 observed with excitation at 405 nm; (c) Fluorescence image of Lysotracker Red with excitation at 543 nm; (d) overlay image of (a), (b), and (c). Figure S17. Confocal fluorescence images of intracellular Cd 2+ in HEK293 cells with DQCd1. (a) HEK 293 cells incubated with DQCd1 (10 µm) at 37 C for 30 min; (b g) DQCd1 stained cells were exposed to 30 µm CdCl 2 at 25 C for 5, 10, 15, 25, 40, 60 min; (h, i) sequestration of intracellular Cd 2+ by addition of 50 µm TPEN for 5, 10 min. (A) Bright-field transmission images. (B) Fluorescence images with emission collected at nm. (C) Fluorescence images with emission collected at nm. (D) Ratio images generated from (B) and (C), F nm /F nm. S11

12 (S1) a) E. S. Kryachko, E. V. LudeÇa, Energy Density Functional Theory of Many Electron Systems, Kluwer Academic Publisher, Dordrecht, 1990; b) W. Koch, M. C. Holthausen, A Chemist s Guide to Density Functional Theory, 2nd ed., Wiley-VCH, Weinheim, (S2) M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford CT, (S3) S. Sumalekshmy, M. M. Henary, N. Siegel, P. V. Lawson, Y. G. Wu, K. Schmidt, J.-L. Bredas, J. W. Perry, C. J. Fahrni. J. Am. Chem. Soc. 2007, 129, (S4) Taki, M.; Desaki, M.; Ojida, A.; Iyoshi, S.; Hirayama, T.; Hamachi, I.; Yamamoto, Y. J. Am. Chem. Soc. 2008, 130, (S5) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Coord. Chem. Rev. 1999, 184, 311. (S6) Martell, A. E., Smith, R. M. Critical Stability Constants, Volume 1: Amino Acids, Plenum Press, New York, (S7) Perrin, D. D.; Dempsy. B. Buffers for ph and Metal Ion Control; John Wiley & Sons: Chapman and Hall: New York, London, (S8) (a) M. Shortreed, R. Kopelman, M. Kuhn, B. Hoyland. Anal. Chem. 1996, 68, (b) W. Lin, L. Yuan, L. Long, C. Guo, J. Feng. Adv. Funct. Mater. 2008, 18, (c) A. Caballero, R. Martınez, V. Lloveras, I. Ratera, J. Vidal-Gancedo, K. Wurst, A. Tarraga, P. Molina, J. Veciana. J. Am. Chem. Soc. 2005, 127, S12

13 NMR spectrum. Figure S15. (a) HMBC spectra (600MHz, DMSO-d 6, 298K) of 1, (b) HMBC spectra (600MHz, DMSO-d 6, 298K) of 1 HCl. Figure S16. (a) Patial 1 H- 1 H COSY spectra (600MHz, DMSO-d 6, 298K) of DQCd1, (b) HMBC spectra (600MHz, DMSO-d 6, 298K) of DQCd1. Figure S17. (a) Patial 1 H- 1 H COSY spectra (600MHz, DMSO-d 6, 298K) of Cd-DQCd1, (b) HMBC spectra (600MHz, DMSO-d 6, 298K) of Cd-DQCd1. S13

14 Figure S18. (a) Patial 1 H- 1 H COSY spectra (600MHz, DMSO-d 6, 298K) of Zn-DQCd1, (b) HMBC spectra (600MHz, DMSO-d 6, 298K) of Zn-DQCd1. S14

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A dominant homolytic O-Cl bond cleavage with low-spin triplet-state Fe(IV)=O formed is revealed in the mechanism of heme-dependent chlorite dismutase

Supplementary Information to: A dominant homolytic O-Cl bond cleavage with low-spin triplet-state Fe(IV)=O formed is revealed in the mechanism of heme-dependent chlorite dismutase Shuo Sun, Ze-Sheng Li,