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1 Electronic upplementary Material (EI) for ChemComm. This journal is The Royal ociety of Chemistry 217 upporting Information A two-photon fluorescent probe for specific detection of hydrogen sulfide based on a familiar EIPT fluorophore bearing AIE characteristics Liyan Chen, a Di Wu, a* Chang u Lim, b Dayoung Kim, a ang-jip am, a Woolin Lee, a Gyungmi Kim, a Hwan Myung Kim, b* and Juyoung Yoon a* a Department of Chemistry and ano cience, EwhaWomans University, eoul, 12-75, Korea b Department of Chemistry and Energy ystems Research, Ajou University, uwon, Korea jyoon@ewha.ac.kr; wudi @163.com; kimhm@ajou.ac.kr Table of Contents 1. General Information ynthesis of Probe Fluorescence Quantum Yield Measurement 4 4. UV-vis spectrum and detection Limit 4 5. Reaction mechanism ph-dependence electivity of probe after addition biothiols and sulfite Response of H 2 in serum 8 9. Two-Photon Fluorescence Microscopy Copies of MR and Mass spectrum References..14 1

2 1. General Information Unless otherwise noted, materials were purchased from commercial suppliers and used without further purification. All the solvents were treated according to general methods. Flash column chromatography was performed using 2-3 mesh silica gel. Chemical shifts were reported on the delta (δ) scale in parts per million (ppm) relative to the singlet ( ppm) for tetramethylsilane (TM). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet), coupling constants (Hz) and integration. 13 C MR spectra were recorded at 75 MHz with complete proton decoupling. Chemical shifts are reported in ppm relative to the central line of the triplet at 77. ppm for CDCl 3. Fluorescence emission pectra were obtained using a RF-531/PC spectrofluorophotometer (himadzu). UV absorption spectroscopy measurements were carried out on cinco -31 using a 1 cm optical path length cell at room temperature. Confocal microscopy experiment was conducted with a confocal laser scanning biological microscope (lympus, FV12, Japan) and super resolution TED laser (stimulated emission depletion) confocal microscopy (Leica TC P8, Germany), excitation source: 45 nm diode laser. The high resolution mass spectra (HRM) were measured on a Bruker Ultraflex Xtreme MALDI-TF/TF mass spectrometer by EI. 2

3 2. ynthetic pathway of probe 1. H CH 3 H H 2 + I 2, MeH HMTA,TFA H CH 3 CH H Imidazole THF/H Probe 1 cheme 1. ynthetic pathway of probe 1. ynthesis of compounds 2 and 3: The intermediates 2 and 3 were synthesized according to the literature 1. ynthesis of probe 1: The compound 3 (67 mg,.25 mmol, 1 equiv.) and imidazole (255 mg,.38 mmol, 1.5 equiv.) were totally dissolved in 4 ml the mixture of tetrahydrofuran and distilled water (v:v = 1:1) under Ar. Then 2-cyclopenten-1-one (41 mg,.5 mmol, 2 equiv.) was added. The mixture was warm to 4 o C. Upon completion of the reaction as monitored by TLC, the final mixture was diluted by 1 M HCl (2 ml) and extracted with ethyl acetate (3 15 ml). The organic layer was concentrated under reduced pressure. After these, chromatography of the crude product on silica gel, using DCM and n-hexane as eluant, gave pure the probe 1, 357 mg (43 %). Probe 1 was synthesized according to the literature 2. Compound 3: δh (3 MHz, CDCl 3 ): (1 H, s), 1.42 (1 H, s), 7.94 (1 H, d, J = 8.1), 7.87 (1 H, d, J = 7.8), 7.77 (1 H, s), 7.62 (1 H, s), (1 H, m), (1 H, m), 2.33 (3 H, s);δc (75 MHz, CDCl 3 ): 19.61, , , , , , 132.3, , , 125.7, , , , , Probe 1: δ H (3 MHz, CDCl 3 ): 8.35 (1 H, s), (1 H, m), (1 H, m), (1 H, m), (1 H, m), 7.26 (1 H, d, J = 2.5), 7.21 (1 H, s), 5.46 (1 H, t, J = 7.8), (1 H, m), (1 H, m), 2.48 (1 H, dd, J = 1.6, 8.9), 2.42 (3 H, d, J = 1.6), 2.38 (1 H, d, J = 12.1);δ C (75 MHz, CDCl 3 ): 21.2, , 152.6, 15.84, 136.9, , , , 132.4, , , , 122.9, , , ,76.28, 37.18, 27.92, 2.46; HRM (EI) m/z = , calcd for [M+H] + C 2 H 16 2 =

4 3. Fluorescence Quantum Yield Measurement Fluorescence quantum yield of probe 1 and probe 1 after additon of 3 equivalents of a 2 was determined in the reference of fluoresceinin.1 M aqueous ah. The quantum yield of probe 1 and probe 1 with a 2 are calculated according to the equation:ф x =Ф s (A s x )/(A x s ), where Ф s is the fluorescence quantum yield of fluorescein (Ф =.98), A x is the absorbance of probe 1 and probe 1 after addition with a 2, respetively. A s is the absorbance of the fluorescein. x is integrated fluorescence emission intensity of probe 1 and probe 1 with a 2 while the s is integrated fluorescence emission intensity of fluorescein. 4. UV-vis spectrum and detection limit. A) Absorbance Probe Probe +.1 EQ H2 Probe +.2 EQ H2 Probe +.3 EQ H2 Probe +.4 EQ H2 Probe +.5 EQ H2 Probe +.6 EQ H2 Probe +.7 EQ H2 Probe +.8 EQ H2 Probe + 1. EQ H2 Probe EQ H2 Probe EQ H2 Probe + 2. EQ H2 Probe EQ H2 Probe + 3. EQ H2 Probe + 4. EQ H Wavelength / nm 4

5 B) 3 Equation y = a + b* Adj. R-quare Intensity of FL at 54 nm Value tandard Error B Intercept B lope H 2 probe Linear Fit of B Concentration / M Fig 1. A) UV-vis spectra of probe (1 μm) in PB buffer (1. mm, ph = 7.4, containing 1% DMF) unpon addtion different concentration of H 2. B) Detection limit of probe 1 response to H 2. pectra were recorded after incubation of H 2 for 3 min. 5. Reaction mechanism of probe reacted with H 2. H H H H Probe 1 Product [M+H] + = [M+H] + = Fig 2. Reaction mechanism of probe 1 and H 2. Procedure: The probe 1 (9. mg,.25 mmol, 1 equiv.) and a 2 (1 mg,.125 mmol, 5. equiv.) were totally dissolved in the mixture of PB (ph = 7.4) and DMF (v:v = 4:1). The reaction was stirred at room temperature for 2 hours. The mixture was diluted and extracted with DCM (3 8 ml). The organic layer was concentrated under reduced pressure. After these, chromatography of the crude product on silica gel, using DCM and n-hexane as eluant, gave pure the product, 7 mg (81 %). 5

6 Product: δ H (3 MHz, CDCl 3 ): (1 H, s), (1 H, m), (2 H, m), (3 H, m), (1 H, m), 5.41 (1 H, d, J = 1.1), 2.68 (2 H, s), (2 H, m), 2.23 (3 H, s), 2.7 (1 H, s);δ C (75 MHz, CDCl 3 ): 27.26, , , 153.1, , , 133.6, , , , 127.3, , , , , , 37.69, 35.8, 26.64, 2.55; HRM (EI) m/z = , calcd for [M+H] + C 2 H = ph-dependence of probe 1 and probe after addition of H 2 8 Probe + 5 eq. a 2 Probe Intensity at 54 nm ph Fig 3. Fluorescence intensity of probe 1 (1 μm) at 54 nm under different ph values in the absence and the presence of 3 equiv. of H 2 in PB buffer (1. mm, ph = 7.4, containing 1% DMF). pectra were recorded after incubation of H 2 for 3 min. 6

7 7. electivity of probe after addition biothiols and sulfite. 8 ( A ) 8 ( B ) 7 7 Intensity of FL at 54 nm Probe (1 M) Probe (1 M) (1 mm) Probe (1 M) + H 2 (3 M) Intensity of FL at 54 nm Probe (1 M) Probe (1 M) + GH (1 mm) Probe (1 M) + H 2 (3 M) Time / econds Time / econds 8 ( C ) 8 ( D ) 7 7 Intensity of FL at 54 nm Probe (1 M) Probe (1 M) + Cys (1 mm) Probe (1 M) + H 2 (3 M) Intensity of FL at 54 nm Probe (1 M) Probe (1 M) + Hcy (1 mm) Probe (1 M) + H 2 (3 M) Time / econds Time / econds Fig 4. Time course experiment of 1 (1 μm) with (A) a 2 3 (1 mm), (B) GH (1 mm), (C) Cys (1 mm) and (D) Hcy (1 mm) in PB buffer (1. mm, ph 7.4, 1% DMF) relative to the time course of 1 (1 μm) with H 2 (3 μm) in the same condition. 8 Probe(1 M)+ubstrate Indicated (1 mm) Probe(1 M)+ubstrate Indicated (1 mm) + H 2 (5 M) I/I Probe nly 3 2- GH CY HCY Fig 5. Fluorescent spectra of probe (1 μm) in PB buffer (1. mm, ph = 7.4, containing 1% DMF) unpon addtion of 1 mm of sulfite, GH, Cys and Hcy. Fluorescent response of H 2 (5 μm) with probe 1 (1 μm) in the presence of these analytes. pectra were recorded after incubation of H 2 for 3 min. 7

8 8. Response of probe after addition of H 2 in two different concentration of serum. FL mg/dl serum Probe in 2 mg/dl serum Probe +.25 EQ H 2 in serum Probe +.5 EQ H 2 in serum Probe +.75 EQ H 2 in serum Probe + 1. EQ H 2 in serum Probe EQ H 2 in serum Probe + 2. EQ H 2 in serum Probe EQ H 2 in serum Probe + 3. EQ H 2 in serum Probe EQ H 2 in serum Probe + 4. EQ H 2 in serum Probe EQ H 2 in serum Probe + 5. EQ H 2 in serum Wavelength / nm Fig 6. Fluorescent spectra of probe 1 (1 μm) in serum (2 mg/ dl) with 1% DMF as co-solvent. Excitation Wavelength = 35 nm. lit width = 5*5 nm. Reaction time = 3 min. FL mg/dl serum Probe in 4 mg/dl serum Probe +.25 EQ H 2 in serum Probe +.5 EQ H 2 in serum Probe +.75 EQ H 2 in serum Probe + 1. EQ H 2 in serum Probe EQ H 2 in serum Probe + 2. EQ H 2 in serum Probe EQ H 2 in serum Probe + 3. EQ H 2 in serum Probe EQ H 2 in serum Probe + 4. EQ H 2 in serum Probe + 5. EQ H 2 in serum Wavelength / nm Fig 7. Fluorescent spectra of probe (1 μm) in serum (4 mg/ dl) with 1% DMF as co-solvent. Excitation Wavelength = 35 nm. lit width = 5*5 nm. Reaction time = 3 min. 8

9 9. Two-Photon Fluorescence Microscopy. Two-photon fluorescence microscopy images of probe 1-labeled cells were obtained with spectral confocal and multiphoton microscopes (Leica TC P2 MP) with 1 oil objectives, numerical aperture (A) = 1.3. The two-photon fluorescence microscopy images were obtained with a DMI6B Microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Chameleon, 9 MHz, 2 fs; Coherent Inc., anta Clara, CA, UA) set at wavelength 74 nm and output power 135 mw, which corresponded to approximately mw/cm 2 average power in the focal plane. To obtain images at 5 6 nm range, internal PMTs were used to collect the signals in an 8 bit unsigned pixels at 2 Hz scan speed, respectively. Flurorescence intensity analysis was carried out using Leica software. 9

10 1. Copies of 1 H MR, 13 C MR and Mass pectra CH H CH H 1

11 11

12 H H 12

13 H H 13

14 11. Reference 1. Z. J. Huang,.. Ding, D. H. Yu, F. H. Huang and G. Q. Feng, Chem. Commun., 214, 5, a) X. Chen,.-K. Ko, M. J. Kim, I. hin and J. Yoon, Chem. Commun., 21, 46, 2751; b) F.-J. Huo, Y.-Q. un, J. u, J.-B. Chao, H.-J. Zhi and C.-X. Yin, rg. Lett., 29, 11, 4918; c) F.-J. Huo, Y.-Q. un, J. u, Y.-T. Yang, C.-X. Yin and J.-B. Chao, rg. Lett., 21, 12, 4756; d) Y.-M. Dong, Y. Peng, M. Dong and Y.-W. Wang, J. rg. Chem., 211, 76,