Low-Affinity Zinc Sensor Showing Fluorescence

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1 Supporting Information for Low-Affinity Zinc Sensor Showing Fluorescence Responses with Minimal Artifacts Xinhao Yan, a Jin Ju Kim, b Hey Sun Jeong, c Yu Kyung Moon, b Yoon Kyung Cho, c Soyeon Ahn, a Sang Beom Jun, c,d* Hakwon Kim, a * and Youngmin You b * a Department of Applied Chemistry, Kyung Hee University, Yongin, Gyeonggi-do 17104, Korea hwkim@khu.ac.kr b Division of Chemical Engineering and Materials Science, Ewha Womans University, Seoul 03760, Korea odds2@ewha.ac.kr c Department of Electronics Engineering and d Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul 03760, Korea juns@ewha.ac.kr CONTENTS Figure S1. 1 H NMR spectrum (300 MHz, CDCl3) of compound 2 S5 Figure S2. 13 C NMR spectrum (75 MHz, CDCl3) of compound 2 S5 Figure S3. 1 H NMR spectrum (300 MHz, CDCl3) of compound 3 S6 Figure S4. 13 C NMR spectrum (75 MHz, CDCl3) of compound 3 S6 Figure S5. 1 H NMR spectrum (300 MHz, CDCl3) of compound 4 S7 Figure S6. 13 C NMR spectrum (75 MHz, CDCl3) of compound 4 S7 Figure S7. 1 H NMR spectrum (300 MHz, CDCl3) of compound 5 S8 Figure S8. 13 C NMR spectrum (75 MHz, CDCl3) of compound 5 S8 Figure S9. 1 H NMR spectrum (300 MHz, CDCl3) of HBO ACR S9 Figure S C NMR spectrum (75 MHz, (CD3)2CO) of HBO ACR S9 Figure S11. 1 H NMR spectrum (300 MHz, CDCl3) of compound 6 S10 Figure S C NMR spectrum (75 MHz, CDCl3) of compound 6 S10 Figure S13. 1 H NMR spectrum (300 MHz, (CD3)2CO) of HBO ACR S11 Figure S C NMR spectrum (75 MHz, (CD3)2CO) of HBO ACR S11 Figure S15. 1 H NMR spectrum (300 MHz, CDCl3) of compound 7 S12 Figure S C NMR spectrum (75 MHz, CDCl3) of compound 7 S12 S1

2 Figure S17. 1 H NMR spectrum (300 MHz, CDCl3) of compound 8 S13 Figure S C NMR spectrum (75 MHz, CDCl3) of compound 8 S13 Figure S19. 1 H NMR spectrum (300 MHz, CDCl3) of HBO ACR S14 Figure S C NMR spectrum (75 MHz, CDCl3) of HBO ACR S14 Figure S21. UV vis absorption (a) and photoluminescence (b) spectra for 10 M HBO ACR (CH3CN) in the absence (black) and presence of 200 equiv Zn(ClO4)2 (blue) or 200 equiv Bu4NOH (red) S15 Figure S22. Comparison of the photoluminescence spectra (normalized to 1) of HBO and HBO ACR in air-equilibrated aqueous buffer solutions containing 25 mm PIPES and 100 mm KCl S15 Figure S23. Figure S24. Table S1. Figure S25. Figure S26. UV vis absorption changes of 20 M HBO ACR recorded with the addition of ZnCl2 (0 50 equiv) (a) Comparison of the experimental (solid lines) and simulated (CPCM(water) TD B3LYP/LANL2DZ:6 311+G(d,p)//B3LYP/LANL2DZ: G(d,p); dotted lines) UV vis absorption spectra for a zinc-free deprotonated form (black) and a zinc-bound form (red) of the model structure of HBO ACR. See the chemical structures in (b) for the input structures. Bars correspond to calculated oscillator strength values. Isodensity (isovalue = 0.02) surface plots for the molecular orbitals that participate in the lowest energy singlet transition (S1) of the deprotonated form (b) and the zinc-bound form (c) Summary of the TD DFT Calculation Results for the Model Compounds Shown in Figures S24b and S24c Fluorescence Job plot of the zinc complexation of HBO ACR (ph 7.4 buffer containing 25 mm PIPES and 100 mm KCl) Determination of the Kd value of HBO ACR in the absence of KCl. (a) Fluorescence spectra ( ex = 347 nm) of 5.0 M HBO ACR with increasing the concentration of ZnCl2, and (b) the corresponding fluorescence zinc titration isotherm (black squares) and a non-linear least-squares fit to a S16 S16 S16 S17 1:1 binding model (red curve) S17 Figure S27. ESI mass (positive mode) spectra for aqueous solutions of 100 M HBO ACR in the presence of (a) 1 mm KCl, (b) 5 mm ZnCl2, and (c) 1 mm KCl + 5 mm ZnCl2 S18 S2

3 Figure S28. Determination of Kd values of HBO and HBO ACR. Fluorescence spectra (top panels, ex = 347 nm) of 5.0 M HBO (a) and 5.0 M HBO ACR (b) with increasing the concentration of ZnCl2 (0 1.0 mm), and corresponding fluorescence zinc titration isotherms (black squares) and a non-linear least-squares fit (red curve) to a 1:1 binding model (bottom panels) S19 Figure S29. Determination of the potassium dissociation constants HBO ACR (a) and the compound 5 (b). Photoluminescence intensities of 5 M HBO ACR or 5 M compound 5 were recorded with increasing the concentrations of KCl (0 4.0 M). The corresponding titration isotherms plotting integrated photoluminescence intensities as functions of the KCl concentration (black squares) were fit to a 1:1 binding model (red curves) S19 Figure S30. Determination of the Kd value of HBO ACR. (a) Fluorescence spectra ( ex = 347 nm) of 5.0 M HBO ACR with increasing the concentration of ZnCl2, and (b) corresponding fluorescence zinc titration isotherms (black squares) and a non-linear least-squares fit to a 1:1 binding model (red curve) S20 Figure S31. UV absorption (a) and photoluminescence spectra (b) of 5 M HBO ACR in the absence (black) and presence of 10 equiv ZnCl2 (red) and after the subsequent addition of 10 equiv TPEN (blue). (c) Corresponding integrated photoluminescence intensities S20 Figure S32. Fluorescent zinc selectivity of 5 M HBO ACR examined in KCl-free buffer solutions (25 mm PIPES, ph 7.4): Blue bars, metal-free state; red bars, in the presence of metal ions; black bars, after subsequent addition of 50 M ZnCl2. Na + and Ca 2+ ions are 1.0 mm. Mg 2+ ion is 100 M S21 Figure S33. Fluorescent zinc selectivity of (a) 5 M HBO and (b) 5 M HBO ACR. Blue bars, metal-free state; red bars, in the presence of metal ions; black bars, after subsequent addition of 50 M ZnCl2 S21 Figure S34. UV vis absorption (a and c) and photoluminescence (b and d) spectra obtained for 50 M HBO ACR (air-equilibrated milli-q water containing 100 mm KCl) in the absence (a and b) and presence (c and d) of 2.5 mm ZnCl2 with decreasing ph from 10.5 to 3.1. (e) Corresponding ph titration isotherm for the zinc-free solution. S22 Figure S35. Photoluminescence decay traces of 10 M HBO ACR (air-equilibrated milli-q water containing 100 mm KCl) obtained at various ph in the absence (empty black circles) and presence (red filled triangles) of 50 equiv ZnCl2 after S3

4 ps pulsed photoexcitation at 345 nm (pulse duration = 8 ps): a, ph 4.1; b, ph 7.4; c, ph 9.9 S22 Table S2. Summary of the Non-linear Least Squares Fit Results of the Decay Traces in Figure S35 S23 Table S3. Photophysical Data for HBO ACR Determined at Various phs S23 Figure S36. Photoluminescence spectra for 5 M HBO ACR in the absence (b) or presence of equiv HCl (a) or 200 equiv Bu4NOH (c) in solvents of different viscosity S24 Figure S37. Intracellular zinc imaging of PANC 1 cells incubated with a 10 M HBO ACR through the 405 nm excitation and 450 nm emission channels: Left, before the treatment of ZnPT; middle, the treatment with 100 M ZnPT (10 min); right, after the subsequent addition of 100 M TPEN (5 min) S24 Figure S38. MTT cell proliferation assay of PANC 1 cells treated with varying concentrations of HBO ACR (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 M; 2 h incubation at 37 C, 5% CO2) S25 Figure S39. Intracellular localization of HBO ACR. PANC 1 cells were treated with 10 M HBO ACR together with various organelle-specific stains: (a) DRAQ5 ( ex = 633 nm; em = nm), nucleus stain; (b) ER Tracker ( ex = 561 nm; em = nm), ER stain; (c) MitoTracker ( ex = 633 nm; em = nm), mitochondrion stain; (d) LysoTracker ( ex = 633 nm; em = nm), lysosome stain. Overlay images of HBO ACR (blue) and organelle-specific stains (other colors) are shown, except the HBO ACR/LysoTracker image which contains the individual images of HBO ACR and LysoTracker. Bottom panels are corresponding 2D-colocalization scatter plots S25 S4

5 Figure S1. 1 H NMR spectrum (300 MHz, CDCl3) of compound 2. Figure S2. 13 C NMR spectrum (75 MHz, CDCl3) of compound 2. S5

6 Figure S3. 1 H NMR spectrum (300 MHz, CDCl3) of compound 3. Figure S4. 13 C NMR spectrum (75 MHz, CDCl3) of compound 3. S6

7 Figure S5. 1 H NMR spectrum (300 MHz, CDCl3) of compound 4. Figure S6. 13 C NMR spectrum (75 MHz, CDCl3) of compound 4. S7

8 Figure S7. 1 H NMR spectrum (300 MHz, CDCl3) of compound 5. Figure S8. 13 C NMR spectrum (75 MHz, CDCl3) of compound 5. S8

9 Figure S9. 1 H NMR spectrum (300 MHz, CDCl3) of HBO ACR. Figure S C NMR spectrum (75 MHz, (CD3)2CO) of HBO ACR. S9

10 Figure S11. 1 H NMR spectrum (300 MHz, CDCl3) of compound 6. Figure S C NMR spectrum (75 MHz, CDCl3) of compound 6. S10

11 Figure S13. 1 H NMR spectrum (300 MHz, (CD3)2CO) of HBO ACR. Figure S C NMR spectrum (75 MHz, (CD3)2CO)) of HBO ACR. S11

12 Figure S15. 1 H NMR spectrum (300 MHz, CDCl3) of compound 7. Figure S C NMR spectrum (75 MHz, CDCl3) of compound 7. S12

13 Figure S17. 1 H NMR spectrum (300 MHz, CDCl3) of compound 8. Figure S C NMR spectrum (75 MHz, CDCl3) of compound 8. S13

14 Figure S19. 1 H NMR spectrum (300 MHz, CDCl3) of HBO ACR. Figure S C NMR spectrum (75 MHz, CDCl3) of HBO ACR. S14

15 Figure S21. UV vis absorption (a) and photoluminescence (b) spectra for 10 M HBO ACR (CH3CN) in the absence (black) and presence of 200 equiv Zn(ClO4)2 (blue) or 200 equiv Bu4NOH (red). ex = 331 nm. Figure S22. Comparison of the photoluminescence spectra (normalized to 1) of HBO and HBO ACR in air-equilibrated aqueous buffer solutions containing 25 mm PIPES and 100 mm KCl. S15

16 Figure S23. UV vis absorption changes of 20 M HBO ACR recorded with the addition of ZnCl2 (0 50 equiv). Condition: air-equilibrated aqueous buffer solutions containing 25 mm PIEPES and 100 mm KCl (ph 7.4). Figure S24. (a) Comparison of the experimental (solid lines) and simulated (dotted lines; CPCM(water) TD B3LYP/LANL2DZ:6 311+G(d,p)//B3LYP/LANL2DZ:6 311+G(d,p)) UV vis absorption spectra for a zinc-free deprotonated form (black) and a zinc-bound form (red) of the model structure of HBO ACR. See the chemical structures in (b) for the input structures. Bars correspond to calculated oscillator strength values. Isodensity (isovalue = 0.02) surface plots for the molecular orbitals that participate in the lowest energy singlet transition (S1) of the deprotonated form (b) and the zinc-bound form (c). Table S1.Summary of the TD DFT Calculation Results for the Model Compounds Shown in Figures S24b and S24c species state transition energy (nm) participating MO (expansion coefficient) oscillator strength zinc-free deprotonated form T1 557 HOMO LUMO (0.69) - S1 410 HOMO LUMO (0.70) T2 394 HOMO 3 LUMO (0.56) - S16

17 T3 357 HOMO 2 LUMO (0.45) HOMO 1 LUMO (0.49) - T4 343 HOMO LUMO+1 (0.69) - S2 342 HOMO 2 LUMO (0.42) HOMO 1 LUMO (0.55) T1 496 HOMO LUMO (0.69) - zinc-bound deprotonated form T2 394 HOMO 2 LUMO (0.62) - S1 378 HOMO LUMO (0.70) T3 359 HOMO 1 LUMO (0.70) - S2 356 HOMO 1 LUMO+1 (0.69) Figure S25. Fluorescence Job plot of the zinc complexation of HBO ACR (ph 7.4 buffer containing 25 mm PIPES and 100 mm KCl). Total concentration was 10 M. Figure S26. Determination of the Kd value of HBO ACR in the absence of KCl. (a) Fluorescence spectra ( ex = 347 nm) of 5.0 M HBO ACR with increasing the concentration of ZnCl2, and (b) the corresponding fluorescence zinc titration isotherm (black squares) and a non-linear leastsquares fit to a 1:1 binding model (red curve). Note that the isotherms are a function of calculated concentrations of free zinc ions ([Zn 2+ ]free). Conditions: air-equilibrated buffer solutions containing 25 mm PIPES (ph 7.4). S17

18 Figure S27. ESI mass (positive mode) spectra for aqueous solutions of 100 M HBO ACR in the presence of (a) 1 mm KCl, (b) 5 mm ZnCl2, and (c) 1 mm KCl + 5 mm ZnCl2. S18

19 Figure S28. Determination of Kd values of HBO and HBO ACR. Fluorescence spectra (top panels, ex = 347 nm) of 5.0 M HBO (a) and 5.0 M HBO ACR (b) with increasing the concentration of ZnCl2 (0 1.0 mm), and corresponding fluorescence zinc titration isotherms (black squares) and a non-linear least-squares fit (red curve) to a 1:1 binding model (bottom panels). Note that the isotherms are a function of calculated concentrations of free zinc ions ([Zn 2+ ]free). Conditions: air-equilibrated buffer solutions containing 25 mm PIPES and 100 mm KCl (ph 7.4). Figure S29. Determination of the potassium dissociation constants HBO ACR (a) and the compound 5 (b). Photoluminescence intensities of 5 M HBO ACR or 5 M compound 5 were recorded with increasing the concentrations of KCl (0 4.0 M). The corresponding titration S19

20 isotherms plotting integrated photoluminescence intensities as functions of the KCl concentration (black squares) were fit to a 1:1 binding model (red curves). Inset figure is the photoluminescence spectral evolution of HBO ACR in response to the added KCl. Figure S30. Determination of the Kd value of HBO ACR. (a) Fluorescence spectra ( ex = 347 nm) of 5.0 M HBO ACR with increasing the concentration of ZnCl2, and (b) corresponding fluorescence zinc titration isotherms (black squares) and a non-linear least-squares fit to a 1:1 binding model (red curve). Note that the isotherms are a function of calculated concentrations of free zinc ions ([Zn 2+ ]free). Conditions: air-equilibrated buffer solutions containing 25 mm PIPES and 100 mm NaCl (ph 7.4). Figure S31. UV absorption (a) and photoluminescence spectra (b) of 5 M HBO ACR in the absence (black) and presence of 10 equiv ZnCl2 (red) and after the subsequent addition of 10 equiv TPEN (blue). Condition: ph 7.4 aqueous buffer containing 25 mm PIPES and 100 mm KCl. ex = 347 nm. (c) Corresponding integrated photoluminescence intensities. S20

Other divalent metal ions are 10 M. Chloride salts were used. ex = 350 nm. Fluorescence spectra are integrated over the range ems = 380 620 nm. Figure S33.

21 Figure S32. Fluorescent zinc selectivity of 5 M HBO ACR examined in KCl-free buffer solutions (25 mm PIPES, ph 7.4): Blue bars, metal-free state; red bars, in the presence of metal ions; black bars, after subsequent addition of 50 M ZnCl2. Na + and Ca 2+ ions are 1.0 mm. Mg 2+ ion is 100 M. Other divalent metal ions are 10 M. Chloride salts were used. ex = 350 nm. Fluorescence spectra are integrated over the range ems = nm. Figure S33. Fluorescent zinc selectivity of (a) 5 M HBO and (b) 5 M HBO ACR. Blue bars, metal-free state; red bars, in the presence of metal ions; black bars, after subsequent addition of 50 M ZnCl2. Na + and Ca 2+ ions are 1.0 mm. Mg 2+ ion is 100 M. Other divalent metal ions are 10 M. Chloride salts were used. Conditions: air-equilibrates, buffered aqueous solutions containing 25 mm PIPES and 100 mm KCl; ex = 350 nm. Fluorescence spectra are integrated over the range ems = nm. S21

22 Figure S34. UV vis absorption (a and c) and photoluminescence (b and d) spectra obtained for 50 M HBO ACR (air-equilibrated milli-q water containing 100 mm KCl) in the absence (a and b) and presence (c and d) of 2.5 mm ZnCl2 with decreasing ph from 10.5 to 3.1. (e) Corresponding ph titration isotherm for the zinc-free solution. The curve is a non-linear least-squares fit to a relationship, absorbance at 374 nm = [HBO ACR]total (A Ka + B [H + ])/([H + ] + Ka), where [HBO ACR]total is the total concentration of HBO ACR, and A and B are proportionality constants. Figure S35. Photoluminescence decay traces of 10 M HBO ACR (ph 4.1 and 9.9, airequilibrated milli-q water containing 100 mm KCl; ph mm PIPES containing 100 mm KCl) S22

obtained at various ph in the absence (empty black circles) and presence (red filled triangles) of 50 equiv ZnCl2 after ps pulsed photoexcitation at 345 nm (pulse duration = 8 ps): a, ph 4.1; b, ph 7.

1 zinc-free 0.88 (1300) 0.45 (5500) 0.59 50 equiv zinc 0.93 (1400) 0.43 (4700) 0.62 7.4 zinc-free 5.0 (4300) 1.1 (1700) 4.7 50 equiv zinc 4.9 (7900) N.A. b 4.9 9.9 zinc-free 4.3 (4100) N.A. b 4.3 50 equiv zinc 4.

23 obtained at various ph in the absence (empty black circles) and presence (red filled triangles) of 50 equiv ZnCl2 after ps pulsed photoexcitation at 345 nm (pulse duration = 8 ps): a, ph 4.1; b, ph 7.4; c, ph 9.9. Table S2. Summary of the Non-linear Least Squares Fit Results of the Decay Traces in Figure S35 a ph 1 (ns, preexponential factor) 2 (ns, preexponential factor) avg (ns) c 4.1 zinc-free 0.88 (1300) 0.45 (5500) equiv zinc 0.93 (1400) 0.43 (4700) zinc-free 5.0 (4300) 1.1 (1700) equiv zinc 4.9 (7900) N.A. b zinc-free 4.3 (4100) N.A. b equiv zinc 4.8 (7500) N.A. b 4.8 a Biexponential decay model was employed, except the indicated data. b Monoexponential decay. c Weighted average lifetime determined using the relationship avg = Ai i 2 / Ai I, where Ai and i are the preexponential factor and time constant, respectively (i = 1 2). In the case of the traces that follow a monoexponential decay model, avg is equal to 1. Table S3. Photophysical Data for HBO ACR Determined at Various phs a abs (nm, log ) b ems (nm) c d avg (ns) e kr (10 6 s 1 ) f knr (10 8 s 1 ) g 328 (2.98), zinc-free 297 (3.05), ph (3.02) 369 (2.88), 50 equiv ZnCl2 301 (2.89), (2.97) 328 (3.02), ph 4.1 zinc-free state 297 (3.08), (3.02) S23

50 equiv ZnCl2 327 (3.00), 297 (3.06), 285 (3.00) 491 0.0098 0.62 16 16 zinc-free state 327 (2.87), 298 (3.02), 457 0.019 4.3 4.3 2.3 ph 9.9 286 (3.00) 368 (2.91), 50 equiv ZnCl2 302 (2.91), 290 (2.

24 50 equiv ZnCl2 327 (3.00), 297 (3.06), 285 (3.00) zinc-free state 327 (2.87), 298 (3.02), ph (3.00) 368 (2.91), 50 equiv ZnCl2 302 (2.91), 290 (2.97) a 5 M in air-equilibrated aqueous buffer solutions containing 25 mm PIPES and 100 mm KCl. b Absorption peak wavelength. c Fluorescence peak wavelength. d Fluorescence quantum yield determined relatively by using 9,10-diphenylanthracene as a standard. e Fluorescence decay lifetimes. f Radiative rate constant. g Non-radiative rate constant. Figure S36. Photoluminescence spectra for 5 M HBO ACR in the absence (b) or presence of equiv HCl (a) or 200 equiv Bu4NOH (c) in solvents of different viscosity. ex = 347 nm. Figure S37. Intracellular zinc imaging of PANC 1 cells incubated with a 10 M HBO ACR through the 405 nm excitation and 450 nm emission channels: Left, before the treatment of S24

25 ZnPT; middle, the treatment with 100 M ZnPT (10 min); right, after the subsequent addition of 100 M TPEN (5 min). Scale bar = 100 m. Figure S38. MTT cell proliferation assay of PANC 1 cells treated with varying concentrations of HBO ACR (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 M; 2 h incubation at 37 C, 5% CO2) Figure S39. Intracellular localization of HBO ACR. PANC 1 cells were treated with 10 M HBO ACR together with various organelle-specific stains: (a) DRAQ5 ( ex = 633 nm; em = nm), nucleus stain; (b) ER Tracker ( ex = 561 nm; em = nm), ER stain; (c) MitoTracker ( ex = 633 nm; em = nm), mitochondrion stain; (d) LysoTracker ( ex = 633 nm; em = nm), lysosome stain. Overlay images of HBO ACR (blue) and organellespecific stains (other colors) are shown, except the HBO ACR/LysoTracker image which contains the individual images of HBO ACR and LysoTracker. Bottom panels are corresponding 2D-colocalization scatter plots. Overlap coefficients: DRAQ5, 0.04; ER Tracker, 0.00; MitoTracker, 0.01; LysoTracker, S25

Investigation of Cation Binding and Sensing by new Crown Ether core substituted Naphthalene Diimide systems

Investigation of Cation Binding and Sensing by new Crown Ether core substituted Naphthalene Diimide systems Electronic Supplementary Material (ESI) for New Journal of Chemistry. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 218 Supplementary Material Investigation