Supporting Information for. Coumarin-derived Cu 2+ Selective Fluorescence Sensor: Synthesis, Mechanisms, and Applications in living cells

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1 Supporting Information for Coumarin-derived Cu 2+ Selective Fluorescence Sensor: Synthesis, Mechanisms, and Applications in living cells Hyo Sung Jung, Pil Seung Kwon, Jeong Won Lee, Jae Il Kim, Chang Seop Hong, Jong Wan Kim, Shihai Yan, Jin Yong Lee,*, Jung Hwa Lee, Taiha Joo,*, and Jong Seung Kim*, Department of Chemistry, Korea University, Seoul , Korea; Department of Laboratory Medicine, Dankook University Hospital, Cheonan , Korea; Department of Chemistry, Institute of Basic Science, Sungkyunkwan University, Suwon , Korea ; Department of Chemistry, POSTECH, Pohang , Korea jinylee@skku.edu (J. Y. Lee); thjoo@postech.ac.kr (T. H. Joo); jongskim@korea.ac.kr (J. S. Kim) Table of Contents 1. Absorption and fluorescence spectra S2 2. Photophysical data S6 3. Fluorescence changes by naked eyes S6 4. Calculation Details S7 5. MTT assay S9 6. Confocal fluorescence images S10 7. FAB-MS data S H-NMR and 13 C-NMR copies S15 9. References of SI S20 S1

2 1. Absorption and fluorescence spectra Absorbance (a) 1 only, Other metals Cu Wavelength (nm) Fluorescence Intensity (a.u.) 1000 (b) only, Other metals Cu 2+ +Other metals Cu Wavelength (nm) Figure S1. (a) Absorption spectra and (b) fluorescence emission spectra of 1 (20 and 5 µm, respectively) with addition of chloride salts of Li +, Rb +, Cd 2+, Cs +, Ag +, Sr 2+, Ba 2+, Zn 2+, Pb 2+, Co 2+, Hg 2+, Mn 2+, Fe 2+, Ni 2+, Cu 2+, Cu 2+ + Other metals (500 µm, respectively) and Na +, K +, Ca 2+ and Mg 2+ (5mM, respectively) in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M) with an excitation at 430 nm. S2

3 Absorbance (a) 2 only, Other metals Cu Wavelength (nm) Fluorescence Intensity (a.u.) (b) 2 only, Other metals Cu Wavelength (nm) Figure S2. (a) Absorption spectra and (b) fluorescence emission spectra of 2 (20 and 5 µm, respectively) with addition of chloride salts of Li +, Rb +, Cd 2+, Cs +, Ag +, Sr 2+, Ba 2+, Zn 2+, Pb 2+, Co 2+, Hg 2+, Mn 2+, Fe 2+, Ni 2+,Cu 2+ (500 µm, respectively) and Na +, K +, Ca 2+ and Mg 2+ (5mM, respectively) in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M) with an excitation at 430 nm. S3

4 (a) 3 only, Other metals Absorbance Wavelength (nm) Fluorescence Intensity (a.u.) (b) 3 only, Other metals Wavelength (nm) Figure S3. (a) Absorption spectra and (b) fluorescence emission spectra of 3 (20 and 5 µm, respectively) with addition of chloride salts of Li +, Rb +, Cd 2+, Cs +, Ag +, Sr 2+, Ba 2+, Zn 2+, Pb 2+, Co 2+, Hg 2+, Mn 2+, Fe 2+, Ni 2+, Cu 2+ (500 µm, respectively) and Na +, K +, Ca 2+ and Mg 2+ (5mM, respectively) in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M) with an excitation at 430 nm. S4

5 Fluorescence Intensity (a.u.) (a)(b)(c)(d)(e) (f) (g)(h) (i) (j) (k) (l) (m) (n)(o) (p) (q) (r) (s) (t) Figure S4. Metal ion selectivity of 1. Bars indicate the fluorescence ratio (430 nm excitation, 479 nm emission). 1 (5 µm) was added to chloride salts of various metal ions ((a) Li +, (b) Rb +, (c) Cd 2+,(d) Sr 2+,(e) Cs +,(f) Ag + (g) Cu 2+ (h) Ni 2+ (i) Ba 2+ (j) Zn 2+ (k) Pb 2+ (l) Co 2+ (m) Hg 2+ (n) Mn 2+ (o) Fe 2+ (500 µm, respectively) (t) Cu 2+ + other metals and (p) Na + (q) K + (r) Ca 2+ and (s) Mg 2+ (5mM, respectively)) in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M) A = A obs - A h [Cu 2+ ] / ([1]+[Cu 2+ ]) Figure S5. Job s plot of 1 vs. Cu 2+ in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M). S5

6 2. Photophysical data Table S1. Photophysical Data α L + Metal ion F 479 Ф f (%) Cu Cu Cu α F 479 : Fluorescence Intensity (a.u.) at 479nm. Ф f : Fluorescence Quantum Yield (fluorescein in 0.1N NaOH as a reference, Ф f = 0.85). S1 3. Fluorescence changes by naked eyes Na + K + Ca 2+ Mg 2+ Li + Cd 2+ Cs + Cu 2+ Ag + Zn 2+ Pb 2+ Hg 2+ Mn 2+ Fe 2+ Ni 2+ Figure S6. Fluorescence responses of 1 (5 µm) in aqueous solution (HEPES:DMSO = 9:1, v/v) (0.01 M) upon the addition of (50 µm) metal ions in water excited at 430 nm using a UV lamp. S6

7 4. Calculational Details To understand the fluorescence quenching behavior of 1 upon addition of Cu 2+, we have carried out ab initio calculations at the density functional theory employing Lee-Yang-Parr three parameterized exchange functionals (B3LYP) with 6-31G* basis sets using a suite of Gaussian 03 programs. S2 Figure S7 shows the calculated frontier molecular orbitals (HOMO, LUMO, LUMO+1, LUMO+2, LUMO+3, and LUMO+4) of 1 and 1-Cu 2+. For both the 1 and 1-Cu 2+, HOMO, LUMO, and LUMO+3 show that the electrons are distributed over the coumarin ring, and LUMO+1 and LUMO+2 show the density over the pyridine ring. On the other hand, LUMO+4 of 1 shows the density over the coumarin ring for 1, while that of 1- Cu 2+ shows the density over the Cu 2+ ion. These orbitals should be related to the fluorescence behavior of 1 upon the complexation as discussed in our previous works. S3 Comparing the frontier molecular orbitals (from HOMO-5 to LUMO+6) of 1 and 1-Cu 2+, though some of them were not shown in Figure S7, we found that LUMO+4 and LUMO+6 of 1-Cu 2+ are the most distinct orbitals as mentioned above. Assuming that the 1-Cu 2+ absorbs a light corresponding to the HOMO-LUMO+4 or HOMO-LUMO+6 energy difference, the electron density shape should change from HOMO to LUMO+4 or LUMO+6, which implies that the electron transfers from the coumarin ring to Cu 2+ ion. This mechanism may cause the fluorescence quenching of 1 upon addition of Cu 2+. LUMO+4 LUMO+4 LUMO+3 LUMO+3 LUMO+2 LUMO+2 S7

8 LUMO+1 LUMO+1 LUMO LUMO HOMO HOMO 1 1-Cu 2+ Figure S7. B3LYP/6-31G* calculated molecular orbitals of 1 and 1-Cu 2+. Table S2. The contribution of each electronic oscillators (orbital transitions) to the lowest energy transition. Electronic oscillators 1 Electronic oscillators 1-Cu 2+ HOMO-5 LUMO 10.78% HOMO LUMO 58.06% HOMO-5 LUMO % HOMO LUMO % HOMO-2 LUMO % HOMO LUMO % HOMO LUMO 67.43% HOMO LUMO % To confirm the contribution of LUMO+4 and LUMO+6 of 1-Cu 2+, we have calculated the excited states calculations using the CIS method using the Hartree-Fock (HF) wavefunctions. The contributions of each electronic oscillators to the lowest energy transition (from the ground state to the first excited state) are listed in Table S2. The electronic oscillators corresponding to the HOMO LUMO+4 and HOMO LUMO+6 transitions are calculated to be 18 and 11%, respectively. The number (29 %) is not so large but these numbers can be modified depending on the calculational method and the basis sets, and such S8

9 a density change may cause the complex not to jump to the fluorescent state. Thus, the fluorescence quenching behavior is mainly due to LUMO+4 and LUMO+6 of 1-Cu 2+ which evidently show the photo-induced electron transfer from the coumarin ring to the Cu 2+ ion. 5. MTT assay only Cu Cu 2+ Cell viability(%) Concentration (µm) Figure S8. Percentage of LLC-MK2 cell viability remaining after cell treatment with 1, Cu 2+ and 1 in the presence Cu 2+ (untreated cells were considered to have 100% survival). Cell viability was assayed by the MTT method (values: mean ± standard deviation). S4 S9

10 6. Confocal fluorescence images Figure S9. Confocal fluorescence images of Cu 2+ in NCTC clone 1469 cells (Zeiss LSM 510 META confocal microscope, 40 objective lens). (a) Bright-field transmission image of NCTC clone 1469 cells incubated with 1 (20 µm). (b) An overlay image of a and c. (c) Fluorescence image of NCTC clone 1469 cells incubated with 1 (20 µm). Further incubated with addition of various concentrations of CuCl 2 [(d) 5, (e) 10 and (f) 20 equiv, respectively]. Figure S10. Confocal fluorescence images of Cu 2+ in LLC-MK2 cells (Zeiss LSM 510 META confocal microscope, 40 objective lens). (a) Bright-field transmission image of LLC- MK2 cells. (b) Fluorescence image of LLC-MK2 cells incubated with 1 (1 µm). Further incubated with addition of various concentrations of CuCl 2 [(c) 5, (d) 10 and (e) 20 equiv, respectively]. (f) Return of intracellular Cu 2+ to the resting level was achieved by addition of EDTA (100 µm). S10

11 (a) (b) Figure S11. (a) Confocal fluorescence 3D image and (b) 3D slice images of LLC-MK2 cell incubated with 1 (5 µm) (Zeiss LSM 510 META confocal microscope, 63 objective lens). S11

12 7. FAB-MS data Mass spectrometric data were obtained on a JEOL-JMS-HX 110A/110A High Resolution Tendem Mass Spectrometry in Korean Basic Science Institute (Korea). Figure S12. FAB-MS of 1. S12

13 Figure S13. FAB-MS of 2. Figure S14. FAB-MS of 3. S13

14 Figure S15. FAB-MS of 1 with CuCl 2. S14

15 8. 1 H NMR and 13 C NMR copies 1 H-NMR and 13 C-NMR spectra were recorded on a VARIAN (200 MHz for 1 H NMR, 50 MHz for 13 C NMR) spectrometer with chemical shifts reported as ppm (in CDCl 3, TMS as internal standard). Figure S16. 1 H NMR spectra (200 MHz) of 1 (10 mm) in CDCl 3. S15

16 Figure S C NMR spectra (50 MHz) of 1 in CDCl 3. S16

17 Figure S18. 1 H NMR spectra (200 MHz) of 2 (10 mm) in CDCl 3. S17

18 Figure S C NMR spectra (50 MHz) of 2 in CDCl 3. S18

19 Figure S20. 1 H NMR spectra (200 MHz) of 3 (10 mm) in CDCl 3. S19

20 Figure S C NMR spectra (50 MHz) of 3 in CDCl References S1 Wu, J.-S.; Liu, W.-M.; Zhuang, X.-Q.; Wang, F.; Wang, P.-F.; Tao, S.-L.; Zhang, X.- H.; Wu, S.-K.; Lee, S.-T. Org. Lett. 2007, 9, 33. S2 Frisch, M. J. et al. Gaussian 03, Revision A1; Gaussian Inc.: Pittsburgh, PA, S3 (a) Cho, E. J.; Moon, J. W.; Ko, S. W.; Lee, J. Y.; Yoon, J.; Nam, K. C. J. Am. Chem. Soc. 2003, 125, (b) Lee, J. Y.; Cho, E. J.; Mukamel, S.; Nam, K. C. J. Org. Chem. 2004, 69, 943. (c) Kim, S. K.; Lee, S. H.; Lee, J. Y.; Lee, J. Y.; Bartsch, R. A.; Kim, J. S. J. Am. Chem. Soc. 2004, 126, (d) Kim, S. K.; Bok, J. H.; Bartsch, R. A.; Lee, J. Y.; Kim, J. S. Org. Lett. 2005, 7, (e) Kim, H. J.; Kim, S. K.; Lee, J. Y.; Kim, J. S. J. Org. Chem. 2006, 71, S4 For a detailed description of MTT assay, see: (a) Cetin, Y.; Bullerman, L. B. J. Agric. Food Chem. 2005, 53, (b) Roy, I.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Bergey, E. J.; Oseroff, A. R.; Morgan, J.; Dougherty, T. J.; Prasad, P. N. J. Am. Chem. Soc. 2003, 125, (c) Andreescu, S.; Sadik, O. A.; McGee, D. W. Chem. Res. Toxicol. 2005, 18, 466. S20