Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

2 Highly Sensitive and Fast Responsive Fluorescence Turn-on Chemodosimeter for Cu 2+ and its Application in Live Cell Imaging Mengxiao Yu, [a] Mei Shi, [a] Zhigang Chen, [a] Fuyou Li,* [a] Xinxin Li, [b] Yanhong Gao, [c] Jia Xu, [a] Hong Yang, [a] Zhiguo Zhou, [a] Tao Yi, [a] and Chunhui Huang* [a] [a] Department of Chemistry & Laboratory of Advanced Materials Fudan University 220 Handan Road, Shanghai (PR China) [b] School of Materials Science and Engineering East China University of Science and Technology 130 Meilong Road, Shanghai (PR China) [c] Hospital of Xinhua Medicine School of Shanghai Jiaotong University 1665 Kongjiang Road, Shanghai (PR China) [

3 Part 1. The supplementary materials, including UV-vis and fluorescence spectra, charge numbers of atoms in 1 and 4, ESI mass spectra, XPS spectrum, fluorescence images, and the result of trypan blue viability test.. Part 2. Colour versions of Figures 2, 5, 7 and 8, Scheme 3, and Table 1 in Text

4 Part 1. The supplementary materials. Figure S1. Absorption spectra of 20 μm 4 upon addition of μm Hg 2+ in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution. Inset: Absorbance at 566 nm as a function of Hg 2+ concentration. Figure S2. Fluorescence response of 20 μm 4 upon addition of μm Hg 2+ in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). Inset: Emission intensity at 591 nm as a function of Hg 2+ concentration.

5 Figure S3. Fluorescence response of 20 μm 4 upon addition of μm Cu 2+ in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). Inset: Emission intensity at 580 nm as a function of Cu 2+ concentration.

6 Figure S4. Fluorescence spectra of 20 µm 4 and 100 µm Cu 2+ upon the addition of EDTA in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). The final concentrations of EDTA were 100 µm and 1mM, respectively. Figure S5. Fluorescence spectra of 20 µm 4 and 40 µm Hg 2+ upon the addition of EDTA in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). The final concentrations of EDTA were 100 µm and 1mM, respectively.

7 Figure S6. The charge numbers of atoms in 4. Figure S7. The charge numbers of atoms in 1.

8 Figure S8. ESI mass spectrum (positive) of 20 µm 4 in the presence of 2 equiv of Hg 2+ in CH 3 CN/ammonium acetate (0.1 M, 3:7, v/v) solution, indicating the formation of 5. Figure S9. ESI mass spectrum (positive) of 20 µm 4 in the presence of 2 equiv of Cu 2+ in CH 3 CN/ammonium acetate (0.1 M, 3:7, v/v) solution, indicating the formation of 6. Figure S10. ESI mass spectrum (positive) of 20 µm 4 in the presence of 0.5 equiv of Hg 2+ in ethanol, indicating the formation of intermediate carbodiimide 7. The solution was nearly colorless and weakly fluorescent, suggesting the molecules of 7 adopt a spirolactam form.

9 Figure S11. Fluorescence spectra of 20 µm 4 upon addition of 100 µm Cu + and Cu 2+ in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). Figure S12. XPS spectrum of 4 with 0.5 equiv. of Cu 2+. The data analysis was carried out by using the RBD AugerScan 3.21 software provided by RBD Enterprises. Binding energies were calibrated by using the containment carbon (C1s = 284.6eV).

10 Figure S13. ESI mass spectrum (positive) of 20 µm 4 in the presence of 0.5 equiv of Cu 2+ in ethanol, indicating the formation of an intermediate 8 with a Cu + and a series of intermediates such as CuL 2, Cu 2 L 2, Cu 3 L 2 (L stands for the ligand 4).

11 Figure S14. Emission intensity at 580 nm of 20 μm 4 in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution vs ph before and after addition of 100 μm Cu 2+ (λ ex = 510 nm). The ph of the solutions was adjusted by addition of 0.2 mol L -1 HCl (or 0.1 mol L 1 NaOH). Figure S15. Absorption spectra of 20 µm 4 upon the addition of 100µM CuCl 2, Cu(NO 3 ) 2, CuSO 4, respectively, suggesting that the anions have no influence on the response of 4 to Cu 2+.

12 Figure S16. Fluorescence spectra of 20 µm 4 upon addition of 0 20 µm Cu 2+ in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution (λ ex = 510 nm). Inset: Emission intensity at 580 nm as a function of Cu 2+ concentration.

13 Figure S17. Absorption spectra of 20 µm 4 in the presence of different metal ions (10 mm for Na +, K +, Mg 2+, Ca 2+, 25 μm for Fe 2+ and Cu +, and 100 µm for other metal ions) in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution. Figure S18. Fluorescence spectra of 20 µm 4 in the presence of different metal ions (10 mm for Na +, K +, Mg 2+, Ca 2+, 25 μm for Fe 2+ and Cu +, and 100 µm for other metal ions) in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution.

14 Figure S19. Confocal fluorescence, brightfield, and overlay images of MCF-7 cells. The cells were supplemented with 0 (a1-a3), 50 (b1-b3), 100 (c1-c3) or 200 (d1-d3) μm CuCl 2 in the growth media for 20 hours at 37 o C, and then were stained with 50 μm 4 for 30 min at 37 o C (λ ex = 543 nm). Figure S20. Cell viability values (%) estimated by trypan blue viability test. MCF-7 cells were cultured in the presence of 50 or 200 μm Cu 2+ for 20 h at 37 C and then incubated with 50 μm 4 for 30 min at 37 o C.

15 Part 2. Colour versions of Figures 2, 5, 7 and 8, Scheme 3, and Table 1 in Text. Figure 2. Distinct difference in Cu 2+ and Hg 2+ sensing behavior of 4 in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution. (a) Time course of 20 µm 4 upon gradual addition of Cu 2+ and Hg 2+. Absorbance at 555 nm (for Cu 2+ ) and 566 nm (for Hg 2+ ) was recorded respectively. Inset shows the fluorescent responses of 4 to 5 equiv. of Cu 2+ and Hg 2+ under 365 nm UV excitation. (b) Color changes of 20 µm 4 in the presence of different concentrations of Cu 2+ or Hg 2+. Figure 5. Time course of the response of 4 to different concentrations of Cu 2+. Absorbance at 555 nm was recorded in 20 μm 4 in CH 3 CN/HEPES (50 mm, ph 7.2, 3:7, v/v) solution.

16 Figure 7. Confocal fluorescence and brightfield images of HeLa cells. (a) Cells stained with 5 μm 4 for 10 min at 25 o C. (b) Cells supplemented with 50 μm CuCl 2 in the growth media for 20 hours at 37 o C and then incubated with 5 μm 4 for 10 min at 25 o C. (c) Brightfield image of cells shown in panel b. The overlay image of (b) and (c) is shown in (d) (λ ex = 543 nm). Figure 8. (a) Two-photon excited fluorescence image of Cu 2+ supplemented HeLa cells stained with 5 μm 4 for 10 min at 25 o C (λ ex = 880 nm). (b) Fluorescence intensity profile across a HeLa cell shown in (a).

17 Table 1. HOMO-1, HOMO, LUMO and LUMO+1 distributions of 1 and 4 calculated by DFT calculations.