Screening and investigation of a cyanine fluorescence probe for simultaneous sensing of glutathione and cysteine with single excitation

Transcription

1 Electronic upplementary Material (E) for ChemComm. This journal is The Royal ociety of Chemistry 214 upporting nformation for creening and investigation of a cyanine fluorescence probe for simultaneous sensing of glutathione and cysteine with single excitation Xu Wang, Jianzheng Lv, Xueying Yao, Yong Li, Fang Huang, Mengmeng Li, Jie Yang, Xiuyun Ruan, Bo Tang* College of Chemistry, Chemical Engineering and Materials cience, Collaborative nnovation Center of Functionalized Probes for Chemical maging, Key Laboratory of Molecular and ano Probes, Ministry of Education, handong ormal University, Jinan 2514, P. R. China * Corresponding author. Tel.: ; Fax: address: tangb@sdnu.edu.cn (B. Tang). 1

2 Contents Part 1. Enlarged figures in the main manuscript 2. Experimental section 3. Molecular structure of biothiols and sulfydryl-containing control compounds 4. DFT calculation results 5. The summarized photophysical properties of the dyes after reaction with biothiols 6. The fluorescence spectra of Cy-3-2 in the presence of biothiols 7. Effect of ph on sensing behavior of Cy electivity investigation of Cy Fluorescence spectra of Cy-3-2 in the presence of sulfydryl-containing control compounds 1. 1 HMR spectra investigation on Cy-3-2 in the presence of β-me 11. M spectra of Cy-3-2 in the presence of sulfydryl-containing control compounds 12. Reaction mechanism of Cy-3-2 towards sulfydryl-containing control compounds 13. M spectra of Cy-3-2 in the presence of biothiols 14. The proposed overall reaction mechanism 15. Colocalization fluorescence images Part 1. The fluorescence spectra of Cy-Ph-Ph in the presence of biothiols 2. Effect of ph on sensing behavior of Cy-Ph-Ph 3. electivity investigation of Cy-Ph-Ph 4. HPLC-M spectra of Cy-Ph-Ph 5. Confocal fluorescence images of Cy-Ph-Ph 2

3 Part 1. Enlarged figures and tables in the main manuscript H Cy-3-2 Cy-4-2 Cy-2,4-2 Cy-Ph-Ph 2 H Cy--Ph Cy--Ph Cy-- 2 Figure 1 Molecular structures of synthesized dyes. Cy--Ph Table 1 Photophysical properties of the synthesized dyes Dyes a abs max /n m a Ex max /n m a Em ma x/nm a Extinction coefficient /1 4 M -1 cm -1 a tokes shift/nm b Φ fl ( 1 2 ) Cy--Ph Cy Cy Cy-2, Cy-Ph-Ph 71, Cy--Ph Cy Cy--Ph Cy-Cys Cy-Hcy Cy-GH a Measured in 5 mm phosphate buffer (ph 7.4, 1% CH 3 C). b Φ fl is the relative fluorescence quantum yield estimated by using CG (Φ=.13 9 ) as a fluorescence standard. 3

4 Figure 2 F variation of Cy-3-2 (1 μm) in the presence of GH. (a) The F recorded after Cy-3-2 reacted with, 2.5, 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 2, 3, 4, 5, 7, 9, and 1 μm GH for 2h. (b) The F recorded after Cy-3-2 reacted with GH (5 μm) for, 2.5, 5, 1, 15, 2, 25, 3, 45, 6, 7, 9, 1, 11, and 12 min. λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. Figure 3 F variation of Cy-3-2 (1 μm) in the presence of Cys. (a) The F recorded after Cy-3-2 reacted with, 5, 1, 15, 2, 3, 4, 5, 6, 8, 1, 15, 2, 3, 4, 5, 6, 7, 8, 9, and 1 μmcys for 2h. (b) The F recorded after Cy-3-2 reacted with Cys (5 μm) for, 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 11, and 12 min. λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. Figure 4 The dependence of F of Cy (1 μm) in the presenceof

5 biologicalthiols(5μm each) upon reaction times. (a) λ ex /λ em =71/85 nm. (b) λ ex /λ em =71/755 nm. 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. Figure 5 The proposed reaction mechanism of Cy-3-2 towards biothiols. Figure 6 Confocal fluorescence images of GH and Cys in Hela cells by Cy

6 (a)-(d), images obtained with band path of 7-74 nm (blue fluorescence, pseudo color, assigned to Cys). (e)-(h), images obtained with band path of 76-8 nm (red fluorescence, assigned to GH). (a), (e), (i), without probe. (b), (f), (j), cells were incubated with 5 μm Cy-3-2 for 15 min. (c), (g), (k), cells were incubated with 5 μm Cy-3-2 for 6 min. (d), (h), (l), the cells were first incubated with EM for 3 min, then incubated with 5 μm Cy-3-2 for 15 min. 37 o C, scale bar = 25 μm, 633 nm laser excitation. 2. Experimental section General Methods. All chemicals and solvents for synthesis were purchased from commercial suppliers (igma-aldrich, Aladdin) and were used without further purification, unless otherwise stated. The composition of mixed solvents is given by the volume ratio (v/v). All synthesis were carried out under an argon atmosphere. Fluorescence spectra were obtained by a FL-92 Edinburgh Fluorescence pectrometer (Edinburgh nstruments Ltd, England) with a Xenon lamp and 1.-cm quartz cells at the slits of 5./5. nm. 1 H-MR and 13 C-MR spectra were taken on a Bruker Advance 3-MHz spectrometer (Bruker, Germany), δ values are in ppm relative to TM. HPLC-HRM analysis was carried out on a Bruker maxis UHR-TF Ultra High Resolution Quadrupole-time of flight mass spectrometer (Bruker Co., Ltd., Germany). The fluorescence images of cells were taken using a TC P5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens ( 2). Design and ynthesis. Heptamethine cyanine chlorine (Cy.7.Cl) was synthesized in our laboratory. 1 To tune the electrophilic reactivity, selectivity, and sensitivity of the dyes towards biological thiols, different -, -, and -aryl substituents as leaving groups were introduced on the heptamethine cyanine moiety, in which chemical bonds of C-, C-, and C- are acted as the activatable sites for thiols. The oxylinking R dyes were obtained by substituting chlorine atom on Cy.Cl.7 with phenol, m-nitrophenol, p-nitrophenol, and -(4-hydroxy-3-nitrophenyl)benzamide (HB) under alkaline conditions to form Cy--Ph, Cy-3-2, Cy-4-2, and Cy-Ph-Ph, respectively. n the case of Cy-2,4-2, 2,4-dinitrofluorobenzene was added to the alkaline solution of ketone cyanine 2 to react for 8 h. The thio-linking R dyes were synthesized by substituting chlorine atom on Cy.Cl.7 with thiophenol and p- nitrothiophenol to form Cy--Ph and Cy-- 2, respectively. The nitrogen-linking R dye of Cy--Ph was obtained by the same procedure by using aniline. However, the reaction between nitroaniline and Cy.Cl.7 gave so low productivity that the pure product could not be obtained. We expected that the nitrobenzene derivatives could serve as good quenchers and the number of nitro group or its different substitution position on benzene ring might result in different reactivity towards thiols, resulting in dramatic fluorescence enhancement or spectral shift. The reaction products of Cy- Cys, Cy-Hcy, and Cy-GH were obtained by incubating Cy-3-2 with excessive amounts of biothiols (Cy-3-2 : biothiols with molar ratio of 1:1) at room temperature for 24h (a time scale that allowed the intramolecular rearrangement 6

7 reaction to complete) and the obtained solutions were applied directly to the test. For the purpose of mechanism investigation, Cy-β-ME was synthesized by nucleophilic substitution on Cy-3-2 by β-mercaptoethanol (β-me). ynthesis of Cy--Ph. Cl phenol TEA, DMF r.t. Cy.Cl.7 Cy--Ph Cy.Cl.7 (.64 g,.1 mmol) and phenol (.188 g, 2. mmol) were dissolved in DMF (3 ml), and triethylamine (1 μl) was added. The reaction mixture was stirred under an argon atmosphere for 24 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (dichloromethane/methanol = 25/1 as eluent) to give Cy- -Ph (45 mg, 65%) as green solid. 1 H MR (d6-dm, 3 MHz): δ 1.26 (s, 18H), 1.96 (s, 2H), 2.52 (s, 4H), 4.2 (d, 4H, J = 13.8 Hz), 6.2 (d, 2H, J = 14.4 Hz), 7.7 (t, 1H, J = 7.2 Hz), (m, 4H), (m, 6H), 7.53 (d, 2H, J = 7.5 Hz), 7.85 (d, 2H, J = 14.4 Hz). 13 C MR (d6-dm, 75 MHz): δ 171.5, , , , 14.98, 14.78, 13.34, , , , 122.3, , , 11.95, 99.89, 48.51, 29.4, 27.3, 23.7, 2.67, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy Cl m-nitrophenol TEA, DMF r.t. Cy.Cl.7 Cy-3-2 Cy.Cl.7 (.64 g,.1 mmol) and m-nitrophenol (.139 g, 1. mmol) were dissolved in DMF (5 ml), and triethylamine (5 μl) was added. The reaction mixture was stirred under an argon atmosphere for 24 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (dichloromethane/methanol = 2/1 as eluent) to give Cy-3-2 (47 mg, 63%) as green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 18H), (m, 2H), (m, 4H), 4.2 (q, 4H, J = 6.9 Hz), 6.26 (d, 2H, J = 14.4 Hz), (m, 2H), 7.37 (q, 4H, J = 3.9 Hz), 7.53 (d, 2H, J = 7.2 Hz), (d, 2H, J = 9. Hz), (m, 2H), (m, 2H). 13 C MR (d6-dm, 75 MHz): δ 171.3, 16.77, , 149.2, , 141.5, 14.6, 132.6, , , , , 12.73, , , 19.33, 1.37, 7

8 48.62, 27.1, 23.69, 2.61, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy Cl p-nitrophenol TEA, DMF r.t. Cy.Cl.7 Cy-4-2 Cy.Cl.7 (.64 g,.1 mmol) and p-nitrophenol (.139 g, 1. mmol) were dissolved in DMF (5 ml), and triethylamine (5 μl) was added. The reaction mixture was stirred under an argon atmosphere for 24 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (dichloromethane / methanol = 2/1 as eluent) to give Cy-4-2 (44 mg, 6%) as green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 18H), (m, 2H), 2.76 (d, 4H, J = 5.4 Hz), 4.19 (q, 4H, J = 6.6 Hz), 6.27 (d, 2H, J = 14.4 Hz), (m, 2H), (m, 4H), 7.47 (d, 2H, J = 9. Hz), 7.54 (d, 2H, J = 7.5 Hz), 7.72 (d, 2H, J = 14.1 Hz), 8.37 (d, 2H, J = 9.3 Hz). 13 C MR (d6-dm, 75 MHz): δ , , 16.58, 142.9, 141.5, 141.5, , , , , , , 12.57, , , 1.4, 48.62, 35.7, 31.23, 29.4, 28.99, 28.78, 28.65, 28.53, 27.2, 26.52, 25.7, 23.67, 22.4, 2.52, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy-2,4-2. Ketone cyanine 2 (.62 g,.1 mmol) and K 2 C 3 (.69 g,.5 mmol) were dissolved in DMF (2 ml), and 2,4-dinitrofluorobenzene (63 μl,.5 mmol) was added. The reaction mixture was stirred under an argon atmosphere for 6 hours at 8 o C. Then the mixture was filtered and the filtrate was concentrated. After that, the crude product was purified through column chromatography over silica (dichloromethane / methanol = 1/3 as eluent) to give Cy-2,4-2 (51 mg, 65%) as green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 18H), 1.94 (br, 2H), 2.73 (d, 4H, J = 26.1 Hz), 4.2 (q, 4H, J = 7.2 Hz), 6.26 (d, 2H, J = 14.4 Hz), (m, 2H), (m, 5H), (m, 4H), 8.43 (d, 1H, J = 9.3 Hz), 9.1 (s, 1H). 13 C MR (d6-dm, 75 MHz): δ , , , , , 8

9 141.2, , , 129.8, , , , , , , , 1.93, 55.98, 54.87, 48.76, 4.33, 4.5, 39.78, 39.5, 39.22, 38.94, 38.66, 38.38, 27.1, 26.84, 23.73, 18.52, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of -(4-hydroxy-3-nitrophenyl)benzamide (HB). 2 H H 2 4-amino-2-nitrophenol benzoyl chloride TEA, DMF r.t. H 2 H C HB 4-amino-2-nitrophenol (.154 g, 1. mmol) was dissolved in DMF (2 ml), and benzoyl chloride (162 μl, 1.4 mmol) in 5 ml dichloromethane was added dropwise at o C. After adding triethylamine (385 μl), the reaction mixture was stirred under an argon atmosphere for 6 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (petroleum ether-dichloromethane gradient elution from 1:1 to 1:3) to give -(4-hydroxy-3-nitrophenyl)benzamide (13 mg, 5%) as orange solid. 1H MR (d6-dm, 3 MHz): δ 7.2 (d, 1H, J = 39 Hz), (m, 4H), (m, 3H), 8.53 (s, 1H), 1.45 (s, 1H). 13 C MR (d6-dm, 75 MHz): δ , , , , , 132.8, 131.7, 131.5, 13.75, 13.5, , , 128.4, , , , , HRM (E - ): m/z calcd for [M-H] - = , found ynthesis of Cy-Ph-Ph. Cl HB TEA, DMF r.t. 2 H Cy.Cl.7 Cy-Ph-Ph Cy.Cl.7 (.32 g,.5 mmol) and -(4-hydroxy-3-nitrophenyl)benzamide (.258 g, 1. mmol) were dissolved in DMF (2 ml), and triethylamine (12 μl) was added. The reaction mixture was stirred under an argon atmosphere for 24 hours at room temperature. Then 5 ml deionized water was added. After the mixture was extracted with dichloromethane (15 ml 3), the obtained organic phase was dried by anhydrous sodium sulfate and filtered. The filtrate was concentrated and was purified through column chromatography over silica (dichloromethane / methanol = 1/3 as eluent) to give Cy-Ph-Ph (28 mg, 65%) as green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 18H), 2.1 (s, 2H), 2.81 (d, 4H, J = 24 Hz), (m, 4H), 6.28 (d, 2H, J = 14.4 Hz), (m, 3H), (m, 4H), (m, 5H), (m, 2H), (m, 3H), 8.75 (s, 1H), 1.59 (s, 1H). 13 C MR (d6-9

10 DM, 75 MHz): δ , , , , , 141.4, , , , , , , , , , , , , , 12.66, 12.12, , 111.5, 1.37, 64.91, 59.64, 48.59, 29.89, 28.86, 26.93, 23.65, 18.54, 13.98, 13.44, LC-HRM (E + ): m/z calcd for [M-] + = , found ; calcd for [M-] 2+ = , found ynthesis of Cy--Ph. Cl thiophenol TEA, DMF r.t. Cy.Cl.7 Cy--Ph Cy.Cl.7 (.54 g,.84 mmol) and thiophenol (.93 μl,.84 mmol) were dissolved in DMF (2 ml), and triethylamine (5 μl) was added. The reaction mixture was stirred under an argon atmosphere for 12 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (dichloromethane-methanol gradient elution from 6:1 to 3:1) to give Cy--Ph (32 mg, 55%) as yellowgreen solid. 1 H MR (d6- DM, 3 MHz): δ (m, 6H), 1.42 (s, 12H), 1.94 (br, 2H), 2.79 (s, 4H), 4.23 (d, 4H, J = 15. Hz), 6.33 (d, 2H, J = 14.4 Hz), 7.13 (t, 1H, J = 5.7 Hz), (m, 1H), 7.56 (d, 2H, J = 7.2 Hz), 8.65 (d, 2H, J = 14.1 Hz). 13 C MR (d6- DM, 75 MHz): δ 171.5, , , , , 136.7, , , , , , , , , 11.43, 59.72, 48.57, 27.7, 25.84, 2.4, 14.6, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy Cl p-nitrothiophenol TEA, DMF r.t. Cy.Cl.7 Cy-- 2 Cy.Cl.7 (.64 g,.1 mmol) and p-nitrothiophenol (.156 g, 1. mmol) were dissolved in DMF (3 ml), and triethylamine (5 μl) was added. The reaction mixture was stirred under an argon atmosphere for 12 hours at room temperature, and then was poured into 2 ml diethyl ether to obtain darkgreen precipitate. After filtration, the solid was redissolved by a small amount of dichloromethane and was purified through column chromatography over silica (dichloromethane / methanol = 5/1 as eluent) to give Cy-- 2 (37 mg, 49%) as green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 18H), 1.98 (s, 2H), 2.82 (s, 4H), 4.25 (d, 4H, J = 14.1 Hz), 6.36 (d, 2H, J = 14.4 Hz), 7.25 (t, 2H, J = 6.3 Hz), (m, 4H), (m, 1

11 4H), 8.2 (d, 2H, J = 9. Hz), 8.51 (d, 2H, J = 14.4 Hz). 13 C MR (d6-dm, 75 MHz): δ 171.7, , , , , , , , , 126.4, , , , , 11.78, 62.76, 48.81, 45.29, 27., 25.86, 12.25, HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy--Ph. Cl Cy.Cl.7 aniline TEA, DM 65 o C H Cy--Ph Cy.Cl.7 (.37 g,.58 mmol) and aniline (56.6 μl,.58 mmol) were dissolved in DM (3 ml), and triethylamine (5 μl) was added. The reaction mixture was stirred under an argon atmosphere for 12 hours at 65 o C. Then 5 ml deionized water was added and the mixture was extracted with ethyl acetate (15 ml 3). The obtained organic phase was dried by anhydrous sodium sulfate and filtered. The filtrate was concentrated and was purified through column chromatography over silica (dichloromethane-methanol gradient elution from 1:1 to 5:1) to give Cy--Ph (27 mg, 67%) as blue-green solid. 1 H MR (d6-dm, 3 MHz): δ (m, 6H), 1.56 (s, 12H), 1.76 (t, 2H, J = 6.6 Hz), 2. (q, 2H, J = 4.5 Hz), 3.52 (s, 6H), 3.97 (t, 4H, J = 4.5 Hz), 5.32 (t, 1H, J = 4.5 Hz), 5.74 (d, 2H, J = 12.9 Hz), (m, 4H), (m, 4H), 7.45 (d, 2H, J = 7.2 Hz). HRM (E + ): m/z calcd for [M-] + = , found ynthesis of Cy-β-ME. 2 H H H TEA, DMF r.t. Cy-3-2 (74 mg,.1 mmol) was dissolved in DMF (5 ml), and β-me (74 μl, 1 mmol) and triethylamine (5 μl) were added. The reaction mixture was stirred under an argon atmosphere for 24 hours at room temperature. Then the solvent was removed under reduced pressure, and the crude product was purified through column chromatography over silica (dichloromethane/ethanol = 2/1 as eluent) to give Cy-β- ME (26. mg, 38%) as green solid. 1 H MR (d6-dm, 3 MHz): δ 1.7 (s, 12H), 1.84 (t, 2H, J = 5.4 Hz), 2.1 (q, 2H, J = 3.7 Hz), 2.68 (t, 4H, J = 6. Hz), 2.9 (t, 2H, J = 6.6 Hz), 3.55 (t, 6H, J = 6.5 Hz), 4.27 (q, 4H, J = 6.5 Hz), 6.33 (d, 2H, J = 14.1 Hz), (m, 2H), (m, 4H), 7.69 (d, 2H, J = 7.5 Hz), 8.82(d, 2H, J = 14.1 Hz). 13 C MR (d6-dm, 75 MHz): δ , , , , , , , , , , 11.25, 72.62, 6.5, 56.37, 49.18, 31.61, 11

12 29.32, 27.65, 26.4, 22.43, 2.96, 18.9, 14.28, HRM (E + ): m/z calcd for [M-] + = , found Fluorescence pectral Measurement. A stock solution (1 mm) of the probe in CH 3 C was prepared. During the measurement, a sample solution was prepared by mixing an appropriate amount of the stock solution of the probe with an appropriate amount of each biothiol and finally diluting with PB buffer (5 mm, ph 7.4) to obtain the desired concentration. The fluorescence intensities of the R dyes were similarly measured with a slit width of 5 nm 5 nm. The detection was performed at 37 o C. Quantum Yield Measurement. Fluorescence quantum yields for the synthesized dyes were determined by using CG (Φ f =.13 in.1 M PB of ph 7.4) as a fluorescence standard. 3 The quantum yield was calculated using the following equation: Φ F(X) = Φ F() (A F X / A X F ) (n X /n ) 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. ubscripts and X refer to the standard and to the unknown, respectively. For the synthesized dyes, the excitation wavelength was at 7 nm while keeping the absorption below.5. Cell Confocal maging Experiment. Hela cells were maintained following protocols provided by the American Type Tissue Culture Collection. Cells were seeded at a density of cells ml -1 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% fetal bovine serum (FB) and 1% antibiotics (penicillin /streptomycin, 1 U ml -1 ). Cultures were maintained in a humidified incubator at 37 C, in 5% C 2 /95% air. Cells were passed and plated on 18-mm glass coverslips in culture dish. A solution of 5 μμ probe in culture solution was prepared before confocal imaging. Excitation of probe-loaded cells at 633 nm was carried out with a Hee laser and the emission was collected between 7-8 nm for Cy-Ph-Ph, and 7-74/76-8 nm for Cy-3-2. Prior to imaging, the medium was removed. Cell imaging was carried out after washing cells with PB buffer (ph 7.4,.1 M) for three times. Colocalization maging Experiments in Hela Cells. Hela cells were passed and dispersed on 24 mm glass coverslips at 37 C, 5% C 2 1 day before imaging. Then cells were incubated with Mito-Tracker Green (5 nm) and Cy-3-2 (5 μμ) for 15 min. The medium was removed and cells were washed with PB (1 mm, ph 7.4) for three times. Confocal images of cells fluorescence of the Mito-Tracker Green were captured using a 488 nm laser, the collection window is nm. The excitation wavelength of Cy-3-2 is 633 nm, and the collection window is 7 8 nm. HPLC-HRM Experiment. Chromatographic separation by HPLC (DEX Ultimate 3) was performed with a C18 column (25 mm 4.6 mm i.d., 5 μm, DEXC18) using a mobile phase composed of 1 HCH and methanol with differing proportions. The flow rate is.3 ml/min and the detection wavelength is 67 nm. The HPLC system was interfaced to the E-M system (maxis UHR-TF 12

13 Ultra High Resolution Quadrupole-time of flight mass spectrometer, Bruker Co., Ltd., Germany). ample of 8. μl were injected into the column using an autosampler. 3. Molecular structure of biothiols and sulfydryl-containing control compounds H H 2 H GH H H H H 2 H H Cys H 2 H H Hcy H H H H H H H 2 β-me AC Cysteamine cheme 1. The molecular structure of biothiols and sulfydryl-containing control compounds. 4. DFT calculation results 4 Table 1. Bond length, bond order, and net charge of central carbon obtained from DFT calculations. Dyes Bond length C-X /Å Bond order C-X et charge of central carbon Cy--Ph (C-).9372 (C-).381 Cy (C-).923 (C-).368 Cy (C-).919 (C-).366 Cy-2, (C-).898 (C-).353 Cy-Ph-Ph (C-).9146 (C-).371 Cy--Ph 1.81 (C-) 1.34 (C-) Cy (C-) (C-) Cy--Ph (C-) (C-)

14 Cy-3-2 Cy-4-2 Cy-2,4-2 Cy-Ph-Ph Cy--Ph Cy--Ph Cy-- 2 Cy--Ph Figure 1. The optimized molecular structure of the synthesized R dyes from DFT calculation. 5. The summarized photophysical properties of the dyes after reaction with biological thiols 14

15 Fluorescence ntensity Fluorescence ntensity Fluorescence ntensity (a) Cy--Ph Control Cys Hcy GH (c) Cy-4-2 (e) Cy-Ph-Ph Control Cys Hcy GH Control Cys Hcy GH Fluorescence ntensity Fluorescence ntensity Fluorescence ntensity 14 (b) Cy Control Cys Hcy GH (d) Cy-2,4-2 Control Cys Hcy GH (f) Cy--Ph Control Cys Hcy GH Fluorescence ntensity (g) Cy-- 2 Control Cys Hcy GH Fluorescence ntensity (h) Cy--Ph Control Cys Hcy GH Figure 2. Fluorescence emission spectra (λex=71 of the synthesized R dyes (1 μm) after reaction with 5 μm of GH, Cys, and Hcy respectively in 5 mm PB buffer (ph=7.4, 1% CH 3 C) at 37 o C for 2h. (a) Cy--Ph, (b) Cy-3-2, (c) Cy-4-2, (d) Cy-2,4-2, (e) Cy-Ph- Ph, (f) Cy--Ph, (g) Cy-- 2, (h) Cy--Ph.

16 Table 2. The photophysical properties of the dyes after reaction with biological thiols. The experimental conditions can be found in the caption of Figure 2. Dyes (λem, Cy--Ph (78 Cy-3-2 (786 Cy-4-2 (788 Cy-2,4-2 (76 Cy-Ph-Ph (783 Cy--Ph (85 Cy-- 2 (86 Cy--Ph (78 ew peak or not (λem, yes (778 yes (755 yes (758 yes (755 Dye+Cys Dye+Hcy Dye+GH ew ew peak peak red/blue red/blue shift red/blue shift F/F ( λ, or not F/F ( λ, or not shift ( λ, (λem, (λem, blue shift (2 blue shift (33 blue shift (3 blue shift ( no.89 yes (755 yes (755 blue shift (5 blue shift (51 yes (776 yes (797 blue shift (4 red shift ( no no yes (797 yes (797 yes (797 red shift (14 blue shift (8 blue shift (9 1.5 yes (795 yes (85 yes (88 yes (82 yes (83 red shift (15 red shift (2 red shift (2 red shift (42 red shift (2 F/F no yes (88 red shift (2 no 1.2 no.92 no The fluorescence spectra of Cy-3-2 in the presence of biothiols A good linearity between the F and the GH concentration in the range of.25-4 μm was obtained (Figure 3b). As to Cys, a good linearity between the F and the Cys concentration in the range of 5.-1 μm was observed (Figure 4b). F85nm (a) Concentration of GH (μm) F85nm (b) F= [GH] R= Concentration of GH (μm) 16

17 Figure 3. Dependence of F of Cy-3-2 (1 μm) upon different GH concentrations. (a) F of Cy-3-2 with, 2.5, 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 2, 3, 4, 5, 7, 9, and 1 GH. (b) The linear relationship between F of Cy-3-2 and GH concentrations. Experimental conditions: 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h, λ ex /λ ex =71/85 nm. F755nm (a) Concentration of Cys (μm) F755nm 3 (b) F= [Cys] R= Concentration of Cys (μm) Figure 4. Dependence of F of Cy-3-2 (1 μm) upon different Cys concentrations. (a) F of Cy-3-2 with, 5, 1, 15, 2, 3, 4, 5, 6, 8, 1, 15, 2, 3, 4, 5, 6, 7, 8, 9, and 1 μm Cys. (b) The linear relationship between F of Cy-3-2 and Cys concentrations. Experimental conditions: 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h, λ ex /λ ex =71/755 nm. The F of Cy-3-2 remained almost unchanged in the Hcy concentration range of ~6 μm (Figure 5a). ubsequently, the F decreased with increasing Hcy concentration, and the Em max red-shifted to 797 nm (Figure 5b). The dynamic investigation showed a rapid falling of the F to the lowest level within the first 1 min (Figure 6a), and then the F slowly increased with increasing incubation time, accompanied by a red shift of Em max to 797 nm. Even though after 2h, the F was still lower than the control (Figure 6b). Fluorescence ntensity (a) Fluorescence ntensity (b) Figure 5. F variation of Cy-3-2 (1 μm) in the presence of different concentrations of Hcy. (a), 1, 4, and 6 μm Cys. (b) 6, 7, 1, 2, 3, 4, 5, 6, 9, and 1 μm Hcy. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h. 17

18 Fluorescence ntensity (a) 1 min Fluorescence ntensity (b) 12 min 1 min Figure 6. The F recorded after Cy-3-2 (1 μm) reacted with Hcy (5 μm) for (a), 1, 2, 4, 6, 8, and 1 min and (b) 1, 15, 2, 25, 4, 5, 6, 7, 8, 1, 11, and 12 min. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. 7. Effect of ph on sensing behavior of Cy-3-2 The investigation on ph (Figure 7) showed that the Cy-3-2 is stable enough in a relatively wide ph range of , and displays the obvious response for GH and Cys simultaneously at ph 7.4. F85nm (c) (a) ph GH Control F755nm Cys Control Figure 7. Effect of ph on the reaction of (a) Cy-3-2 and GH (λ ex/em =71/85 and (b) Cy- 3-2 and Cys (λ ex/em =71/755. The concentrations of Cy-3-2 and tested biothiols were 1 μm and 5 μm, respectively. 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h. (b) (d) ph 8. electivity investigation of Cy-3-2 To evaluate the specific response of Cy-3-2 to GH and Cys simultaneously, various species were examined in parallel under the same conditions, including amino acids, glucose, GG, ascorbic acid, H 2 2,, and 2 - (Figure 8). t can be seen that the fluorescence of Cy-3-2 apparently enhanced upon reaction with GH or Cys in the respective detection channel. While the fluorescence variations induced by 18

19 other bio-substances was neglectable. These results suggest that Cy-3-2 is highly selective for the targeting biothiols. F 85 nm (a) Cy F755 nm (b) Cy Figure 8. Fluorescence intensity changes of (a) Cy-3-2 (1 μm, λ ex/em =71/85 and (b) Cy-3-2 (1 μm, λ ex/em =71/755 upon addition of various amino acids and biologically related substances after 2h in 5 mm PB buffer (ph=7.4, 1% CH 3 C) at 37 o C. (1) probe only, (2) Gly, (3) Ala, (4) Arg, (5) Asn, (6) Asp, (7) Gln, (8) Glu, (9) His, (1) Val, (11) Leu, (12) Lys, (13) Met, (14) Pro, (15) er, (16) Thr, (17) Trp, (18) Tyr, (19) le, (2) Glucose (5 μm), (21) Ascorbic acid (5 μm), (22) GG (5 μm), (23) (25 μm), (24) - 2 (5 μm), (25) H 2 2 (5 μm), (26) Cys (5 μm), (27) Hcy (5 μm), (28) GH (5 μm). The concentrations of the tested amino acids were all 5 μm. 9. Fluorescence spectra of Cy-3-2 in the presence of sulfydryl-containing control compounds Fluorescence ntensity (a) Control -ME-2h AC-2h Cysteamine-2h Fluorescence ntensity (b) Control Cysteamine-2h

20 Fluorescence ntensity (c) Cysteamine-24h Figure 9. The F recorded after Cy-3-2 (1 μm) reacted with 5 μm β-me, AC, and cysteamine. (a) The overall F after reaction for 2h. (b) The enlarged F of the control and Cy-3-2 +cysteamine after 2h. (c) The F of Cy-3-2 +cysteamine after 24h. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C HMR spectra investigation on Cy-3-2 in the presence of β-me We performed 1 H MR spectral analysis of Cy-3-2 in the presence of β-me (Figure 1). n the presence of 1.5 equiv β-me, the vinylic protons of Cy-3-2 were markedly shifted to downfield regions. The shift of H a at 7.98 ppm gradually disappeared and a new shift at 8.82 ppm emerged, while the shift of H b at 6.26 ppm gradually disappeared with a new emerged shift at 6.33 ppm. n the same time, the proton of o-position in the nitrophenol was shifted to an upfield region (H c 7.7 to 7.58 ppm) owing to the release of free m-nitrophenol. For confirmation, the spectrum of an authentic Cy-β-ME (Figure 1G) was listed and high coincidence can be found between the product (Figure 1F) and Cy-β-ME. The MR spectral variations suggested that the nitrophenol of Cy-3-2 was readily substituted with a thiol group, which is accordance with the fluorescence changes (Figure 1). 2

21 H a H c H H H a H b H b G F E D C B A H a H c H b ppm Figure 1. Partial 1 H MR spectra of Cy-3-2 (15 mg) in the presence of β-me (1.5 equiv) in d6-dm. (A) Cy-3-2, (B).5 h after addition of ME, (C) 1 h, (D) 2 h, (E) 6 h, (F) 24 h, (G) Cy-β-ME. 11. M spectra of Cy-3-2 in the presence of sulfydryl-containing control compounds More detailed information can be obtained from HRM. n the presence of β-me or AC, the peaks assigned to the thiol-substituted products of 6/7 (the numbered compound can be found in the proposed reaction mechanism in Figure 12) were found, respectively (Figure 11a and 11b). However, in the case of cysteamine, a bridging product of (Cy) 2 -cysteamine (1) besides the thiol/amino substituted product (8/9) was observed (Figure 11c and 11d), indicating an intermolecular nucleophilic substitution in the presence of thiol containing amino acids (Table 3). Therefore, based on the F spectra, MR spectra, and HRM spectra, the proposed reaction mechanism of Cy-3-2 upon control compounds addition was shown in Figure 12, exhibiting the intramolecular rearrangement cascade reaction and the subsequent intermolecular nucleophilic coupling reactions. 21

22 ntens. x (a) (6) +M,.2min #1 H (Cy-3-2 ) ntens. x (b) (7) H +M,.2min #12 H m/z m/z ntens. x (c) (1) H +M,.1min #4 (Cy-3-2 ) ntens (d) (8/9) H 2 or +M,.1min #4 H H m/z m/z Figure 11. Mass spectra recorded after Cy-3-2 (1 μm) incubated with sulfydryl-containing control compounds (5 μm) in a 5 mm PB buffer (ph=7.4) at 37 o C. (a) β-me, 2h incubation; (b) AC, 2h incubaiton; (c) and (d) cysteamine, 24h incubation. Table 3. The molecular formula and m/z of compounds in Figure 11. Compounds molecular formula m/z obsd m/z cald 6 C 36 H 45 2 [M-] [M-] C 39 H [M-] [M-] /9 C 36 H 46 3 [M-] [M-] C 7 H [M-2] [M-2] Reaction mechanism of Cy-3-2 towards sulfydryl-containing control compounds 22

23 2 beta-me/ac 6 H or H H 7 Cy-3-2 Cysteamine H 2 8 or H H 9 2 H 1 Figure 12. The proposed mechanism of Cy-3-2 in the presence of sulfydryl-containing control compounds. 13. M spectra of Cy-3-2 in the presence of biothiols ntens (a) (5a) HC H +M,.2min #11 ntens (b) (2a/4a) H 2 CH or +M,.2min #11 H CH H m/z m/z 23

24 2 ntens (c) (5b) HC H +M,.2min #11 ntens. 8 6 (d) (2b/4b) CH H or +M,.2min #11 H CH 2 H m/z m/z ntens. 15 (e) (1) H H 2 H H +M,.1min #8 H m/z Figure 13. Mass spectra recorded after Cy-3-2 (1 μm) incubated with (a) and (b) 5 μm Cys; (c) and (d) 5 μm Hcy; (e) 5 μm GH in a 5 mm PB buffer (ph=7.4) at 37 o C for 2h. Table 4. The molecular formula and m/z of compounds in Figure 13. Compounds molecular formula m/z obsd m/z cald 5a C 71 H [M-2] [M-2] a/4a C 37 H [M-] [M-] b C 72 H [M-2] [M-2] b/4b C 38 H [M-] [M-] C 44 H [M-] [M-]

25 14. The proposed overall reaction mechanism H H d-pet - X ( 2 )m X =, m =, 1, 2 GH Cys (n=1) Hcy (n=2) H 2 inert G emission at longer wavelength 1 CH HC * n=1, 4a n=2, 4b emission at shorter wavelength n emission at longer wavelength n=1, 2a n=2, 2b H n n=1, 3a n=2, 3b HC H n H ( 2 )m HC H n - X n=1, 5a n=2, 5b Figure 14. The proposed overall reaction mechanism of the synthesized R dyes towards biothiols. 15. Colocalization fluorescence images The subcellular localization of Cy-3-2 with Mito Tracker Green (Figure 15) gave a Pearson's correlation coefficient of.41, indicating that Cy-3-2 does not localize to mitochondria. Figure 15. Colocalization fluorescence imaging of Cy-3-2 in Hela cells. (a) Mito-Tracker Green (5 nm, λex=488 nm, collected in the range of (b) Cy-3-2 (5 μm, λex =633 nm, collected in the range of 7 8. (c) Merged images of (a) and (b). (d) Brightfield image. cale bar = 25 μm. 25

26 Part 1. The fluorescence spectra of Cy-Ph-Ph in the presence of biothiols The F monitoring revealed that in the tested GH concentration range (~1 μm), the F of Cy-Ph-Ph increased with increasing concentration of GH, accompanied by a red shift of Em max to 83 nm (Figure 16a). A good linearity between the F and the GH concentration in the range of.25-1 μm was observed (Fig. 17b). n the tested time period (~12 min), the F of Cy-Ph-Ph increased with increasing reaction times in the presence of GH and the Em max steadily red-shifted to 83 nm (Figure 16b). The F of Cy-Ph-Ph in the presence of Cys were recorded (Figure 18). As can be seen from Figure 18, the F of Cy-Ph-Ph at 783 nm increased with Cys concentration up to 7 μm (Figure 18a). However, when more Cys was added, the F did not rise but decreased and kept unchanged after addition of 7 μm Cys (Figure 18b). The dynamic investigation displayed an increase of Cy-Ph-Ph s F within the first 2 min (Figure 19a). After that, the F decreased along the reaction times (Figure 19b). As can be seen, no matter from the point of concentration and dynamics, the F tend to return to where it was original. n the case of Hcy, the F of Cy-Ph-Ph increased with increasing Hcy concentration up to 5 µm, accompanied by a gradual red shift of Em max to 797 nm. After that, the F remained unchanged with more Hcy (Figure 2a). The dynamic examination exhibited a continuing increase of F and a redshift of Em max to 797 nm during the tested reaction period (~12 min, Figure 2b). Compared with GH, the F variation caused by both Cys and Hcy during the experimental period can be neglectful. To further testify the specific response of Cy-Ph-Ph to GH, the F of Cy-Ph-Ph at 83 nm (Em max ) was monitored in the presence of GH, Cys, and Hcy at different incubation times (Figure 21). From Figure 21, it can be concluded that Cy-Ph-Ph possessed the highest / ratio for GH and the influence from Cys/Hcy was tiny. o we expected that Cy-Ph-Ph would be a good candidate as an imaging probe for GH in cells. Fluorescence ntensity 55 5 (a) Fluorescence ntensity (b) 12 min

27 Figure 16. F variation of Cy-Ph-Ph (1 μm) in the presence of GH. (a) The F recorded after Cy-Ph-Ph reacted with, 2.5, 5, 1, 2, 3, 5, 6, 7, 8, 1, 15, 2, 3, 4, 5, 6, 7, 8, 9, and 1 μm GH for 2h. (b) The F recorded after Cy-Ph-Ph reacted with GH (5 μm) for, 2.5, 5, 1, 15, 2, 25, 3, 45, 6, 7, 9, 1, 11, and 12 min. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. F83nm (a) Concentration of GH (μm) F83nm (b) F= [GH] R= Concentration of GH (μm) Figure 17. Dependence of F of Cy-Ph-Ph (1 μm) upon different GH concentrations. (a) F of Cy-Ph-Ph with, 2.5, 5, 1, 2, 3, 5, 6, 7, 8, 1, 15, 2, 3, 4, 5, 6, 7, 8, 9, and 1 μm GH. (b) The linear relationship between F of Cy-Ph-Ph and GH concentrations. Experimental conditions: 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h, λ ex /λ ex =71/83 nm. Fluorescence ntensity (a) Fluorescence ntensity 16 (b) Figure 18. F variation of Cy-Ph-Ph (1 μm) in the presence of different concentrations of Cys. (a), 5, 1, 2, 3, 4, and 5 μm Cys. (b) 7, 1, 2, 3, 4, 5, 6,9, and 1 μm Cys. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h. 27

28 Fluorescence ntensity (a) 2 min Fluorescence ntensity (b) 2 min 12 min Figure 19. The F recorded after Cy-Ph-Ph (1 μm) reacted with Cys (5 μm) for (a), 5, 1, and 2 min and (b) 2, 3, 4, 6, 1, and 12 min. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. 2 Fluorescence ntensity (a) 1 Fluorescence ntensity (b) 12 min Figure 2. F variation of Cy-Ph-Ph (1 μm) in the presence of Hcy. (a) The F recorded after Cy-Ph-Ph reacted with, 5, 1, 2, 4, 6, 9, 1, 2, 5, 8, and 1 μm Hcy for 2h. (b) The F recorded after Cy-Ph-Ph reacted with Hcy (5 μm) for, 1, 2, 5, 7, 9, 11, and 12 min. Experimental conditions: λ ex =71 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. F83nm Cys Hcy GH Time (min) 28

29 Figure 21. The dependence of F of Cy-Ph-Ph (1 μm) in the presence of biological thiols (5 μm each) upon reaction times. Experimental conditions: λ ex /λ ex =71/83 nm, 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C. To gain a better insight into the mechanism of high specificity of Cy-Ph-Ph to GH, density function theory (DFT) calculations were used to optimize the structures of compounds of Table 1 (not included Cy-GH, Cy-Cys, and Cy-Hcy) at the B3LYP/6-31G(d) level, using a suite of Gaussian 9 programs. The results (Table 1) of the calculations show that the bond order of Cy-Ph-Ph (.9146) is apparently smaller than that of Cy-3-2 (.923), meaning its easier attack by sulfydryl. But Cy-Ph-Ph only showed unique specificity to GH. We think the unique structure of HB could be the main source determining the reactivity towards GH. HB holds one amide unit and one nitro group, and GH contains two amides and one primary amine and has longer molecular skeleton in comparison to Cys and Hcy. 5 Therefore, intermolecular hydrogen bond (for example -H ), may form between Cy-Ph-Ph and GH. Moreover, the electrostatic interaction between the indolium cation of Cy-Ph-Ph and the carbonate anion of GH may be another reason contributing to the mutually close interaction. 5 All of these intermolecular interactions will result in a closer distance of the two reactive sites (H group and C- group), resulting in a higher reactivity towards GH. Figure 22 gives the probable interaction between Cy-Ph-Ph and GH. H H H (1) H C H 2 C C - (3) H (2) - + H H Fig. 22. The probable conformation of interaction between Cy-Ph-Ph and GH. nteraction sites (1) and (2) denote hydrogen bond, and site (3) denotes electrostatic interaction. 2. Effect of ph on sensing behavior of Cy-Ph-Ph The investigation on ph (Figure 23) showed that Cy-Ph-Ph are stable enough in a relatively wide ph range of , and display the obvious response for biothiols at ph

30 8 7 GH Control 6 F83nm Figure 23. Effect of ph on the reaction of Cy-Ph-Ph and GH (λ ex/em =71/83. The concentrations of probes and tested biothiols were 1 μm and 5 μm, respectively. 5 mm PB buffer (ph=7.4, 1% CH 3 C), 37 o C incubation for 2h. ph 3. electivity investigation of Cy-Ph-Ph To evaluate the specific response of Cy-Ph-Ph to GH, various species were examined in parallel under the same conditions, including amino acids, glucose, GG, ascorbic acid, H 2 2,, and 2 - (Figure 24). t can be seen that the fluorescence of Cy-Ph-Ph showed significant enhancement upon reaction with GH, while the fluorescence variations induced by other bio-substances was neglectable. These results suggest that Cy-Ph-Ph is highly selective for the targeting biothiols. F83 nm (b) Cy-Ph-Ph Figure 24. Fluorescence intensity changes of Cy-Ph-Ph (1 μm, λ ex/em =71/83 upon addition of various amino acids and biologically related substances after 2h in 5 mm PB buffer (ph=7.4, 1% CH 3 C) at 37 o C. (1) probe only, (2) Gly, (3) Ala, (4) Arg, (5) Asn, (6) Asp, (7) Gln, (8) Glu, (9) His, (1) Val, (11) Leu, (12) Lys, (13) Met, (14) Pro, (15) er, (16) Thr, (17) Trp, (18) Tyr, (19) le, (2) Glucose (5 μm), (21) Ascorbic acid (5 μm), (22) GG (5 μm), (23) (25 μm), (24) - 2 (5 μm), (25) H 2 2 (5 μm), (26) Cys (5 μm), (27) Hcy (5 μm), (28) GH (5 μm). The concentrations of the tested amino acids were all 5 μm. 4. HPLC-M spectra of Cy-Ph-Ph 3

31 We investigated the interaction between Cy-Ph-Ph and the tested biothiols by HPLC-HRM, intending to explain its high specificity to GH. The HPLC chromatogram of Cy-Ph-Ph showed a peak at 2.9 min (Figure 25a), while that of mixture solution of Cy-Ph-Ph and GH gave a new peak at 11.6 min (Figure 25b). When applied to M analysis, the peaks at 11.6 and 2.9 min possessed m/z of and , assigning to compound 1 and unreacted Cy-Ph-Ph, respectively (Figure 25c). However, in the presence of Cys (Figure 26) or Hcy (Figure 27), no apparent peak was found except two tiny peaks at 59.1 min (Figure 26b) and at 33.3 min (Figure 27b), which were analyzed and found no information. The results suggested that Cy-Ph-Ph is not so active to Cys and Hcy, which is accorded with the results from fluorescence analysis (Figure 2e). ntens. [mau] 4 (a) Cy-Ph-Ph 2.9 min Time [min] _RD2_1_165.d: UV Chromatogram, 67 nm ntens. [mau] 2 15 (b) Cy-Ph-Ph+GH 11.6 min 2.9 min Time [min] _RE1_1_169.d: UV Chromatogram, 67 nm ntens (c) Cy-Ph-Ph+GH 11.6 min (1) [M-] min (Cy-Ph-Ph) [M-] Time [min] _RE1_1_169.d: EC All M _RE1_1_169.d: EC All M Figure 25. HPLC-HRM spectra of Cy-Ph-Ph in the presence of GH. Cy-Ph-Ph (1 μm) was incubated with 5 μm GH in a 5 mm PB buffer (ph=7.4) at 37 o C for 2h. Then the solution was applied to the chromatographic separation and analysed. (a) Cy-Ph-Ph only, (b) Cy-Ph-Ph + GH, (c) M analysis of (b). Eluant of HCH (1 ): methanol=2:8. 31

32 ntens. [mau] 2 (a) Cy-Ph-Ph ntens. [mau] Time [min] _RE1_1_175.d: UV Chromatogram, 67 nm (b) Cy-Ph-Ph+Cys Time [min] _RE2_1_174.d: UV Chromatogram, 67 nm Figure 26. HPLC spectra of Cy-Ph-Ph in the presence of Cys. Cy-Ph-Ph (1 μm) was incubated with 5 μm Cys in a 5 mm PB buffer (ph=7.4) at 37 o C for 2h. Then the solution was applied to the chromatographic separation. (a) Cy-Ph-Ph only, (b) Cy-Ph-Ph + Cys. Eluant of HCH (1 ): methanol=25:75. ntens. [mau] (a) Cy-Ph-Ph ntens. [mau] Time [min] _RD2_1_165.d: UV Chromatogram, 67 nm (b) Cy-Ph-Ph+Hcy Time [min] _RE1_1_167.d: UV Chromatogram, 67 nm Figure 27. HPLC spectra of Cy-Ph-Ph in the presence of Hcy. Cy-Ph-Ph (1 μm) was incubated with 5 μm Hcy in a 5 mm PB buffer (ph=7.4) at 37 o C for 2h. Then the solution was applied to the chromatographic separation. (a) Cy-Ph-Ph only, (b) Cy-Ph-Ph + Hcy. Eluant of HCH (1 ): methanol=2:8. 32

33 5. Confocal fluorescence images of Cy-Ph-Ph After Hela cells were incubated with 5 μm Cy-Ph-Ph for 15 min, strong red fluorescence was observed (Figure 28), indicating good cell membrane permeability of Cy-Ph-Ph. Then a control experiment was performed by first removing intracellular thiols using 1 mm -methylmaleimide (EM, a thiol-blocking reagent) 6 and then incubating with 5 μm Cy-Ph-Ph. A marked fluorescence quenching was observed (Figure 28e), indicating that Cy-Ph-Ph could be applied for GH imaging in living systems. (a) (b) (c) (d) (e) (f) Figure 28. Confocal fluorescence images of GH in Hela cells by Cy-Ph-Ph. (b) Hela cells were incubated with 5 μm Cy-Ph-Ph for 15 min. (e) The cells were first incubated with EM for 3 min, then incubated with 5 μm Cy-Ph-Ph for 15 min. (a) and (d) Brightfield images of cells in (b) and (e). (c) and (f), merging of bright field images and dark field images. mages were obtained with band path of 7-8 nm upon 633 nm excitation. 37 o C, scale bar = 25 μm. References 1 B. Tang, L. Zhang and K. H. Xu, Talanta, 26, 68, (a) X. Wang, J. un, W. H. Zhang, X. X. Ma, J. Z. Lv and B. Tang, Chem. ci., 213, 4, ; (b) Z. Q. Guo,. W. am,. Park and J. Yoon, Chem. ci., 212, 3, E. asaki, H. Kojima, H. ishimatsu, Y. Urano, K. Kikuchi, Y. Hirata, T. agano, J. Am. Chem. oc., 25, 127, The calculations were carried out using Gaussian 9. The molecules were optimized in the gas phase at B3LYP/6-31G(d) level. Harmonic frequency analysis calculations were performed at the same level to verify the optimized structures to be minima. 33

34 Cite this work as: Gaussian 9, Revision B.1, M. J. Frisch, G. W. Trucks, H. B. chlegel, G. E. cuseria, M. A. Robb, J. R. Cheeseman, G. calmani, V. Barone, B. Mennucci, G. A. Petersson, H. akatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. zmaylov, J. Bloino, G. Zheng, J. L. onnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. shida, T. akajima, Y. Honda,. Kitao, H. akai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. gliaro, M. Bearpark, J. J. Heyd, E. Brothers, K.. Kudin, V.. taroverov, R. Kobayashi, J. ormand, K. Raghavachari, A. Rendell, J. C. Burant,.. yengar, J. Tomasi, M. Cossi,. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. tratmann,. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. chterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. alvador, J. J. Dannenberg,. Dapprich, A. D. Daniels,. Farkas, J. B. Foresman, J. V. rtiz, J. Cioslowski, and D. J. Fox, Gaussian, nc., Wallingford CT, J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J. H. Ryu and J. Yoon, J. Am. Chem. oc., 214, 136, C. Yellaturu, M. Bhanoori and. eeli, J Biol. Chem., 22, 277,