A Unique Approach to Development of Near-infrared Fluorescent Sensors for in Vivo Imaging

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1 Supporting Information for A Unique Approach to Development of ear-infrared Fluorescent Sensors for in Vivo Imaging Lin Yuan, Weiying Lin, * Sheng Zhao, Wensha Gao, Bin Chen, Longwei He, Sasa Zhu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 4182, P. R. China weiyinglin@hnu.edu.cn S1

2 Table of Contents Pages Materials and instruments S3 Determination of the fluorescence quantum yield S3 DFT calculations S3 Synthesis S4 Figure S1 S4 Scheme S1-5.S5-7 Figure S2 S7 Figure S3 S7 Figure S4 S8 Figure S5 S8 Figure S6 S8 Figure S7 S9 Figure S8 S9 Figure S9 S9 Scheme S6 S1 Figure S1 S1 Figure S11 S1 Figure S12 S11 Figure S13 S11 Scheme S7 S11 Figure S14 S12 Figure S15 S12 Figure S16 S13 Figure S17 S13 Table S1.S13 Figure S18-2.S14 Figure S21-23.S15 Scheme S8.S15 Figure S24 S16 Table S2-3 S17 Figure S25 S17 Table S4-5.S18 Figure S26 S18 Table S6-7 S19 Figure S27 S19 Table S8-9..S2 Figure S28 S2 Table S1-11.S21 Figure S29 S21 Figure S3-31 S22 Figure S32 S23 Toxicity studies of IR-H 2 2 and Figure S33..S23 Figure S34-36 S24 Figure S37-38 S25 Fluorescence Imaging using IR-Thiol and Figure S39-4.S26 Toxicity studies of IR-Thiol and Figure S41 S27 S2

3 Materials and instruments: Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. Twice-distilled water was used throughout all experiments. Low resolution mass spectra were performed using an LCQ Advantage ion trap mass spectrometer from Thermo Finnigan or Agilent 11 HPLC/MSD spectrometer. High-resolution electronspray (EI-HRMS) mass spectra were obtained from Bruker APEX IV-FTMS 7.T mass spectrometer. High-resolution mass spectra (ESI-HRMS) analyses were measured on a Finnigan MAT 95 XP spectrometer. MR spectra were recorded on an IVA-4 or BRUKER 5 spectrometer, using TMS as an internal standard. Electronic absorption spectra were obtained on a LabTech UV Power spectrometer; Photoluminescent spectra were recorded with a HITACHI F46 fluorescence spectrophotometer. Cells imaging was performed with a ikon Eclipse TE3 or lympus inverted microscope. The in vivo (living mice) imaging was carried out using a FMT 25 LX Quantitative Tomography In Vivo Imaging System. TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel (mesh 2 3), both of which were obtained from the Qingdao cean Chemicals. Determination of the fluorescence quantum yield: 1-3 Fluorescence quantum yield was determined by using ICG (Φ f =.13 in DMS) for 1a-e or Rhodamine 6G (Φ f =.95 in water) for 1f-g as a fluorescence standard. 1-3 The quantum yield was calculated using the following equation: Φ F(X) = Φ F(S) (A S F X / A X F S ) (n X /n S ) 2 Where Φ F is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission curve, and n is the refractive index of the solvents used. Subscripts S and X refer to the standard and to the unknown, respectively. For compounds 1a-e and ICG, the excitation wavelength was at 67 nm while keeping the absorption below.5; For compounds 1f-g and Rhodamine 6G, the excitation wavelength was at 47 nm while keeping the absorption below.5. DFT calculations. 4 The ground state structures of dyes 1a-g were optimized using DFT with B3LYP functional and 6-31G(d) basis set. The initial geometries of the compounds were generated by the GaussView software. The excited state related calculations (UV-vis absorption) were carried out with the time dependent DFT (TDDFT) with the optimized structure of the ground state (DFT/6-31G(d)). The emission of the fluorophores was calculated based on the optimized S 1 excited state geometry. All of these calculations were performed with Gaussian 9 (Revision A.2). References 1. Valeur, B. Molecular Fluorescence: Principles and Applications, Wiley-VCH, Magde, D.; Rojas, G. E.; Seybold, P. Photochem. Photobiol. 1999, 7, ushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; agano, T. J. Am. Chem. Soc. 21, 132, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; akatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; akajima, T.; Honda, Y.; Kitao,.; akai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; gliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K..; Staroverov, V..; Kobayashi, R.; ormand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,.; Austin, A. J.; Cammi, R.; Pomelli, C.; chterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas,.; Foresman, J. B.; rtiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc., Wallingford CT, GAUSSIA 9 (Revision A.2), Gaussian, Inc., Pittsburgh, PA, 29. S3

4 H 4 Synthesis of compound 4. Resorcin 3a (86. mg,.78 mmol) and ah (6% in mineral oil, 19.2 mg,.78 mmol) were placed in a flask containing anhydrous DMF (3. ml), and the mixture was stirred at room temperature under nitrogen atmosphere for 1 min. Compound 2a (2.1 mg,.31 mmol) in DMF (1. ml) was introduced to the mixture via a syringe, and the reaction mixture was stirred at 25 for 2 h. Diethyl ether (35 ml) was added to this solution, and the solid was collected by filtration. The crude product was purified by silica gel flash chromatography using petroleum ether/ch 2 Cl 2 /MeH (25:25:1) as eluent to give compound 4 as a dark green solid (143.9 mg, yield 65.2%). 1 H MR (4 MHz, CDCl 3 ) δ 8.11 (s, 1H), (d, J = 14. Hz, 2H), (t, J = 8. Hz, 2H), (2H), (t, J = 7.2 Hz, 2H), (m, 2H), (m, 2H), (dd, J = 8., 2. Hz, 1H), (dd, J = 8., 2. Hz, 1H), (d, J = 14. Hz, 2H ), (q, J = 7.2 Hz, 4H), (t, J = 6. Hz, 4H ), (m, 2H), (18H); 13 C MR (1 MHz, CDCl 3 ) 171.5, , 16.51, , , , , 13.15, , , , , , 11.74, 11.2, 15.25, 13.9, 99.28, 49.1, 39.39, 28.5, 27.82, 24.38, 21.8, 12.27; MS (API-ES): m/z = M +. HRMS (ESI) m/z calcd for C 4 H (M + ): Found S H 4% fuming sulfuric acid H S 3 H 1c 1h Synthesis of Compound 1h. Compound 1c (.3 g,.59 mmol) was added carefully to 4% fuming sulfuric acid (1 ml) at 5 o C. The mixture was stirred at -5 o C for 5 min and kept at 2-25 o C. After 1 h, it was poured onto 1 g of crushed iced and mixed carefully, and then the aqueous phase is extracted with dichloromethane/meh (5 1 ml, V/V 5: 1). The organic layers were collected, dried over a 2 S 4, and evaporated under reduced pressure. The blue solid is purified by column chromatography on silica gel flash chromatography using CH 2 Cl 2 /EtH (1: 1). The desired product is obtained as an blue solid (.21 g, yield : 57%). 1 H MR (4 MHz, d 6 -DMS, TMS) δ (s, 1H), 8.96 (d, J = 13.6 Hz, 1H), (2H), 7.57 (d, J = 7.6 Hz, 1H), (2H), 6.84 (d, J = 8.4 Hz, 1H) 6.43 (d, J = 13.6 Hz, 1H), 3.85 (s, 3H), (4H), 1.84 (2H), 1.79(s, 6H). 13 C MR (1 MHz, d 6 -DMS) , 16.33, , 15.3, , , , , , , , , 115.9, , , 112.6, 14.39, 5.38, 28.14, 26.21, 23.54, 2.1. MS (ESI - ) S4

5 H H 1 H 2 H 9 H 1 H 8 H 7 H H 3 11 H 5 H 4 H 6 H 13 H 15 H14 H 12 1c Figure S1. Representative 2D CSY MR spectra (5 MHz) of compound 1c in CDCl 3 at 25 o C. Assignments and proton connectivity of 1c were based on the vicinal and long-distance J-coupling in CSY experiments. All the connections between adjacent protons were observed. H 5 (δ = 8.8, d, J = 13. Hz, 1H) and H 6 (δ = 5.6, d, J = 13.5 Hz, 1H) at the vinyl bridge were identified by the J-coupling constants of the trans double bond. The single peak at 6.58 and 7.32 ppm were assigned to H 1 [which has a long-distance J-coupling with H 2 (δ = 6.78, dd, J = 9., 1.5 Hz)] and H 4, respectively. The correlated proton resonances at 6.78 and 7.21 ppm were assigned to H 2 and H 3, respectively. The correlated proton resonances at 6.84, 7.5, and ppm were assigned to H 1, H 8, and (H 7, and H 9 ), respectively. The single peak at 1.67 and 3.36 ppm were easily assigned to H 11 and H 12, respectively. The proton resonances at (m, 2H) can be attributed to H 14, which has a correlation to proton resonances at 2.62 and 2.67 ppm (H 13 and H 15 ). 2a + 3a alkali, 25 o C, 2h alkali, H 2, 5 o C, 2h 4 1a Scheme S1. Formation of compound 4 from treatment of compound 2a with compound 3a; Conversion of compound 4 to compound 1a. S5

6 Cl 5 o C, alkali, H 2 retro-knoevenagel Cl H 2a chloro-substituted "half" cyanine enol Scheme S2. Formation of chloro-substituted "half" cyanine enol from cyanine 2a via a retro-knoevenagel reaction. H Cl Basic conditions H 2 H H-H Cl H Cl Basic conditions Cl H Cl + Fischer's base Scheme S3. A proposed mechanism for the formation of chloro-substituted "half" cyanine enol from cyanine 2a via a retro-knoevenagel reaction. H Cl + H H 5 o C, alkali, H 2 chloro-substituted "half" cyanine enol 3a 1a Scheme S4. Treatment of chloro-substituted "half" cyanine enol with resorcin 3a in the presence of a base at 5 could not afford product 1a. S6

7 R Fisher aldehyde 5 (commercial available) + R 8 R H + R H R Scheme S5. Retrosynthesis analysis for the novel class of IR dyes exemplified by 1a, 1c, and 1e. Figure S2. 1 H MR spectrum (in CDCl 3 ) of an equivalent mixture of compound 1c synthesized from the different routes shown in Scheme 1 and Scheme 3. Figure S3. 13 C MR spectrum (in CDCl 3 ) of an equivalent mixture of compound 1c synthesized from the different routes shown in Scheme 1 and Scheme 3. S7

8 Figure S4. 1 H MR spectrum (in CDCl 3 ) of an equivalent mixture of compound 1d synthesized from the different routes shown in Scheme 1 and Scheme 3. Figure S5. 13 C MR spectrum (in CDCl 3 ) of an equivalent mixture of compound 1d synthesized from the different routes shown in Scheme 1 and Scheme 3. a) ormalized absorption b) ormalized emission Figure S6. ormalized absorption (a) and emission (b) spectra of compound 1c in ph 7.4 PBS/MeH (1: 1) synthesized from the different routes shown in Scheme 1 ( ) and Scheme 3 ( ). S8

9 a) ormalized absorption b) ormalized emission Figure S7. ormalized absorption (a) and emission (b) spectra of compound 1d in ph 7.4 PBS/MeH (1: 1) synthesized from the different routes shown in Scheme 1 ( ) and Scheme 3 ( ). Fl. intensity (a.u.) Fl. intensity (a. u.) ph Figure S8. ph-dependence of the fluorescence intensity of compound 1a (1 μm) excited at 69 nm with the arrow indicating the change of the fluorescence intensities with ph increase from 2.3 to 7.4. Inset: Fluorescence responses (fluorescence intensity at 716 nm) of compound 1a (1 μm) to different ph values (2.3 to 1.3). 8 Fl. intensity (a.u.) Figure S9. ph-dependence of the fluorescence intensity of compound 1a (1 μm) excited at 645 nm with the arrow indicating the change of the fluorescence intensities with ph enhancement from 2.3 to 7.4. S9

10 H R 1 R 2 H H R 1 R 2 1a: R 1 =Et,R 2 =H; 1c: R 1 =Me,R 2 =H; 1e: R 1 =Et,R 2 =Cl; Scheme S6. ph-dependent equilibrium between the phenolic and phenolate forms of dyes 1a, 1c, and 1e. 1. Absorption Figure S1. ph-dependence of the absorption spectra of compound 1c (1 μm) with the arrows indicating the change of the absorption intensities with ph enhancement from 3. to 9.. a) Fl. intensity (a. u.) Fl. intensity (a. u.) ph b) Fl. intensity (a. u.) Figure S11. a) ph-dependence of the fluorescence intensity of compound 1c (1 μm) excited at 69 nm with the arrow indicating the change of the fluorescence intensities with ph increase from 3. to 7.4. Inset: Fluorescence responses (fluorescence intensity at 718 nm) of compound 1c (1 μm) to different ph values (3. to 9.). b) ph-dependence of the fluorescence intensity of compound 1c (1 μm) excited at 65 nm with the arrow indicating the change of the fluorescence intensities with ph enhancement from 3. to 9.. S1

11 Absorption Figure S12. ph-dependence of the absorption spectra of compound 1e (5 μm) with the arrows indicating the change of the absorption intensities with ph enhancement from 2.3 to 7.4. a) 1 Fl. intensity (a.u.) Fl. intensity (a. u.) ph b) Fl. intensity (a.u.) Figure S13. a) ph-dependence of the fluorescence intensity of compound 1e (5 μm) excited at 69 nm with the arrow indicating the change of the fluorescence intensities with ph increase from 2.3 to 7.4. Inset: Fluorescence responses (fluorescence intensity at 718 nm) of compound 1e (5 μm) to different ph values (2.3 to 8.). b) ph-dependence of the fluorescence intensity of compound 1e (5 μm) excited at 65 nm with the arrow indicating the change of the fluorescence intensities with ph enhancement from 2.3 to 7.4. R R R H H H - H + Ionic form Ionic form eutral form 1f: R = H 1g: R = CH 3 Scheme S7. ph-dependent equilibrium between the neutral and the ionic forms of push-pull chromophores 1f-g. S11

12 A).3 Absorption B) 25 C) 5 Fl. intensity (a. u.) Fl. intensity (a. u.) Figure S14. (A) ph-dependence of the absorption spectra of compound 1f (1 μm) with the arrows indicating the change of the absorption intensities with ph decrease from 6. to 2.3. (B-C) ph-dependence of the fluorescence spectra of compound 1f (1 μm) excited at 454 nm (B) or at 576 nm (C) with the arrows indicating the change of the fluorescence intensities with ph decrease from 6. to 2.3. Absorption a) Fl. intensity (a. u.) b) Figure S15. a) ph-dependence of the absorption spectra of compound 1g (1 μm) with the arrows indicating the change of the absorption intensities with ph decrease from 7. to 3.. b) ph-dependence of the fluorescence spectra of compound 1g (1 μm) excited at 51 nm with the arrows indicating the change of the fluorescence intensities with ph decrease from 7. to 3.. S12

13 1. Absorption Figure S16. Absorption spectra of compounds 1a ( ), 1c ( ), and 1e ( ) in neat newborn calf serum (without co-organic solvent). 4 Fl. intensity (a. u.) Figure S17. Fluorescence emission spectra of compounds 1a ( ), 1c ( ), and 1e ( ) (5 μm) in neat newborn calf serum (without addition of any co-organic solvent). Table S1. Photophysical data of the dyes 1a, 1c, 1e, and 1h in pure newborn calf serum (without addition of any co-organic solvent). λ abs /nm a λ em /nm b Φ f c 1a c e h a The maximal absorption of the dye; b The maximal emission of the dyes; c Φ f is the relative fluorescence quantum yield estimated by using ICG (Φ f =.13 in DMS). S13

14 A) B) C) (D) ormalized intensity (E) ormalized intensity (F) ormalized intensity Figure S18. (Top) Fluorescence images of HeLa cells incubated with compounds 1a (A), 1c (B), or 1e (C). (Bottom) in situ (in cellulo) fluorescence emission spectra of compounds 1a (D), 1c F), or 1e (F) measured in HeLa cells excited at 67 nm and the emission spectra collected between 7 and 85 nm. ormalized intensity ormalized intensity Figure S19. ormalized excitation (left) and emission spectra (right) of compound 1a in ph 7.4 PBS/ THF (1: 1) ( ) and HeLa cells ( ). ormalized intensity ormalized intensity Figure S2. ormalized excitation (left) and emission spectra (right) of compound 1c in ph 7.4 PBS/ THF (1: 1) ( ) and HeLa cells ( ). S14

15 ormalized intensity ormalized intensity Figure S21. ormalized excitation (left) and emission spectra (right) of compound 1e in ph 7.4 PBS/ THF (1: 1) ( ) and HeLa cells ( ). Scheme S8. Synthesis of compound 1h. 3 S H 1c 4% fuming sulfuric acid H S 3 H 1h 12 Fl. intensity (a.u.) Figure S22. Emission spectrum of compound 1h in ph 7.4 PBS (without addition of any co-organic solvent). Fl. intensity (a.u.) Figure S23. Emission spectrum of compound 1h in newborn calf serum (without addition of any co-organic solvent). S15

16 Figure S24. Frontier molecular orbital plots of 1a (phenolate form) (A), and 1b (B) in MeH involved in the vertical excitation (i.e., UV/Vis/IR absorption, left column) and emission (right column). The vertical excitation related calculations are based on the optimized geometry of the ground state (S ), and the emission related calculations were based on the optimized geometry of the excited state (S 1 ). Excitation and radiative processes are marked as solid lines and the non-radiative processes are marked by dotted lines. S16

17 Table S2. Representative C-C ( or ) Bond Lengths of 1a (phenolate form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Table S3. Representative C-C ( or ) Bond Lengths of 1b Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Figure S25. DFT optimized structures and molecular orbital plots (LUM and HM) of 1a (phenolic form) in MeH. In the ball-and-stick representation, carbon, nitrogen, and oxygen atoms are colored in gray, blue, and red, respectively. S17

18 Table S4. Representative C-C ( or ) Bond Lengths of 1a (phenolic form) Determined by DFT Calculations. C-C Bond Length C-/ Bond Length Bond Bond C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Figure S26. DFT optimized structures and molecular orbital plots (LUM and HM) of 1c (phenolate form) and 1d in MeH. In the ball-and-stick representation, carbon, nitrogen, and oxygen atoms are colored in gray, blue, and red, respectively. Table S5. Representative C-C ( or ) Bond Lengths of 1c (phenolate form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C S18

19 Table S6. Representative C-C ( or ) Bond Lengths of 1d Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Table S7. Representative C-C ( or ) Bond Lengths of 1e (phenolate form) Determined by DFT Calculations. C-//Cl Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C C6-Cl Figure S27. DFT optimized structures and molecular orbital plots (LUM and HM) of 1f (eutral form and ionic form) in MeH. In the ball-and-stick representation, carbon, nitrogen, and oxygen atoms are colored in gray, blue, and red, respectively. S19

20 Table S8. Representative C-C ( or ) Bond Lengths of 1f (eutral Form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Table S9. Representative C-C ( or ) Bond Lengths of 1f (Ionic Form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Figure S28. DFT optimized structures and molecular orbital plots (LUM and HM) of 1g (eutral form and ionic form) in MeH. In the ball-and-stick representation, carbon, nitrogen, and oxygen atoms are colored in gray, blue, and red, respectively. S2

21 Table S1. Representative C-C ( or ) Bond Lengths of 1g (eutral Form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Table S11. Representative C-C ( or ) Bond Lengths of 1g (Ionic Form) Determined by DFT Calculations. C-/ Bond Bond Length C1-C C8-C C C2-C C9-C C C3-C C11-C C C4-C C12-C C C7-C C13-C Experimental excitation wavelength (nm) Y = X R = f (Ionic form) 1f-g (eutral form) 1b, 1d, 1a/1c/1e (phenolic form) 1g (Ionic form) 1e (phenolate form) 1a, 1c (phenolate form) Theoretical excitation wavelength (nm) Figure S29. Experimental versus theoretical excitation/absorption wavelengths of the dyes 1a-g. S21

22 .1.8 Absorption Figure S3. Absorption spectra of IR-H 2 2 (1 μm) in ph 7.4 phosphate buffer (containing 1% DMS as a cosolvent) in the absence ( ) or presence ( ) of H 2 2 (2 equiv.). A) Fl. intensity (a. u.) B) 16 Fl. intensity (a. u.) Time, min Figure S31. (A) Fluorescence response of IR-H 2 2 (1 μm) to H 2 2 (1 mm) in newborn calf serum (without addition of any co-organic solvent) with excitation at 67 nm. The spectra were recorded after incubation of the sensor IR-H 2 2 with H 2 2 for to 6 min at room temperature. (B) The fluorescence intensity of IR-H 2 2 (1 μm) in the absence ( ) or presence ( ) of H 2 2 (1 mm) in newborn calf serum solution (without addition of any co-organic solvent). The fluorescence intensities at 733 nm were monitored at time intervals. S22

23 A) B) C) ormalized intensity 75 8 Figure S32. (A-B) Fluorescence images of HeLa cells incubated with IR-H 2 2 (5 μm) before (A) and after (B) addition of H 2 2 (5 μm). (C) ormalized in situ (in cellulo) fluorescence emission spectrum ( ) of IR-H 2 2 (5 μm) in HeLa cells after addition of H 2 2 (5 μm) excited at 67 nm. For comparison, the normalized emission spectrum of IR-H 2 2 (5 μm) in ph 7.4 PBS /THF (1: 1) is also shown ( ). Toxicity studies of IR-H 2 2 : RAW264.7 macrophage cells were grown in MEM (modified Eagle s medium) supplemented with 1% FBS (fetal bovine serum) in an atmosphere of 5% C 2 and 95% air at 37 o C. Immediately before the experiments, the cells were placed in a 96-well plate, followed by addition of increasing concentrations of IR-H 2 2 (99% MEM and 1% DMS). The final concentrations of the sensor were kept from 5 to 15 μm (n = 3). The cells were then incubated at 37 C in an atmosphere of 5% C 2 and 95% air at 37 o C for 24 h, followed by MTT assays. Untreated assay with MEM (n = 3) was also conducted under the same conditions. 1 % Viable Cells a b c d e f 9 h Figure S33. Cytotoxicity of IR-H 2 2 in RAW264.7 macrophage cells. The cells were incubated with IR-H 2 2 at corresponding concentrations (a: μm; b: 5 μm; c: 1 μm; d: 25 μm; e: 6 μm; f: 8 μm; g: 1 μm; h: 15 μm) for 24 h. S23

24 Figure S34. Fluorescence images of RAW macrophages cells stimulated with PMA (3. μg/ml) and co-stained with IR-H 2 2, and MitoTracker Green for 6 min: (a) fluorescence image from green channel (mitochondria staining); (b) fluorescence image from red channel; and c) overlay of the green and red channels..6 Absorption Figure S35. Absorption spectra of IR-Thiol (1 μm) in PBS buffer (5 mm, ph 7.4, containing 3% CH 3 C as a cosolvent) in the absence ( ) or presence ( ) of Cys (1 equiv.). Fl. intensity (a.u.) A) Fl. intensity (a.u.) B) Time, second Figure S36 A) Reaction-time profiles of IR-Thiol (1 μm) in the absence ( ) or presence of Cys [15 μm ( ), 25 μm ( ), 5 μm ( )]. The Fluorescence intensities were continuously monitored at time intervals in ph 7.4 PBS/CH 3 C (7: 3). Excitation at 69 nm. Emission at 716 nm. B) The fluorescence spectra of IR-Thiol (1 μm) in ph 7.4 PBS/CH 3 C (7: 3) for hour ( ), 1 hour ( ), 24 hours ( ), and 48 hours ( ). For comparison, the fluorescence spectrum of IR-Thiol (1 μm) in the presence of 5 μm Cys ( ) for 3 min is also shown. S24

25 12 Fl. intensity (a. u.) Figure S37. Fluorescence intensity (at 716 nm) of IR-Thiol (1 μm) in ph 7.4 PBS/CH 3 C (7: 3) with excitation at 69 nm in the presence of various species. 1, blank; 2, Phe; 3, Gly; 4, Arg; 5, Lys; 6, Tyr; 7, Leu; 8, Glucose; 9, Ser; 1, Val; 11, Hcy, 12, GSH; 13, Cys. 25 Fl. intensity (a. u.) Figure 38. Fluorescence response of IR-Thiol in pure newborn calf serum (without addition of any co-organic solvent) with excitation at 67 nm. The spectra were recorded after addition of IR-Thiol into newborn calf serum solution for to 6 min at room temperature. S25

26 HeLa Cells Incubation and Fluorescence Imaging using IR-Thiol: HeLa cells were cultured in Dulbecco s Modified Eagle Medium (DMEM) supplemented with 1% FBS (fetal bovine serum). ne day before imaging, the cells were seeded in 24-well flat-bottomed plates in an atmosphere of 5% C 2, 95% air at 37 C. Immediately before the experiments, the cells were washed with PBS buffer. Subsequently, the cells were incubated with sensor IR-Thiol (5 µm) in PBS (containing 1% CH 3 C as a cosolvent) for 3 min at 37 ºC, and then washed with PBS three times. For the control experiment, the cells were pretreated with a PBS solution containing -ethylmaleimide (1 mm) for 3 min, and then incubated with sensor IR-Thiol (5 µm) in PBS (containing 1% CH 3 C as a cosolvent) for 3 min at 37 ºC. Fluorescence imaging was then carried out after washing the cells with PBS. Fluorescence imaging of intracelluar thiols was conducted using a ikon eclipase TE3 inverted fluorescence microscope. The microscope settings (brightness, contrast, and exposure time) were held constant before and after pretreatment of cells with -ethylmaleimide to compare the relative intensity of intracellular thiol fluorescence. Figure S39. Bright field and fluorescence images of live HeLa cells. A) Bright field image of HeLa cells incubated with IR-Thiol (5 μm) for 3 min at 37 C; B) Fluorescence image of A); C) Bright-field image of HeLa cells pretreated with -ethylmaleimide (1 mm) for 3 min, and then incubated with IR-Thiol (5 μm) for 3 min at 37 C; D) Fluorescence image of C); E) Average fluorescence intensities of (a) the cells incubated with only IR-Thiol or (b) the cells pre-treated with -ethylmaleimide and then incubated with IR-Thiol. The data were analyzed by software EZ-C1 3.9 FreeViewer. ormalized intensity (A) (B) Figure S4. (A) Fluorescence image of HeLa cells incubated with IR-Thiol (5 μm). (B) ormalized in situ (in cellulo) fluorescence emission spectrum of IR-Thiol (5 μm) ( ) in HeLa cells excited at 65 nm. For comparison, the normalized emission spectrum of 1a in ph 7.4 PBS/THF (1: 1) is also shown ( ). S26

27 Toxicity studies of IR-Thiol: Bel 772 cells were grown in MEM (modified Eagle s medium) supplemented with 1% FBS (fetal bovine serum) in an atmosphere of 5% C 2 and 95% air at 37 o C. Immediately before the experiments, the cells were placed in a 96-well plate, followed by addition of increasing concentrations of IR-Thiol (99% MEM and 1% DMS). The final concentrations of the probe were kept from 5 to 1 μm (n = 4). The cells were then incubated at 37 C in an atmosphere of 5% C 2 and 95% air at 37 o C for 24 h, followed by MTT assays. Untreated assay with MEM (n = 3) was also conducted under the same conditions. 1 % Viable Cells a b c d e f Figure S41. Cytotoxicity of IR-Thiol in Bel 772 cells. The cells were incubated with IR-Thiol at corresponding concentrations (a: μm; b: 5 μm; c: 1 μm; d: 25 μm; e: 5 μm; f: 1 μm) for 24 h. S27

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