A Phenylselenium-Substituted BODIPY Fluorescent Turn-off Probe for

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A phenyl-selenium-substituted BODIPY fluorescent turn-off probe for fluorescence imaging of hydrogen sulfide in living cells Deyan Gong, Xiangtao Zhu, Yuejun Tian, Shi-Chong Han, Min Deng, Anam Iqbal, Wei-Sheng Liu, Wenwu Qin, and Huichen Guo Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 13, 2017

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Analytical Chemistry

A phenyl-selenium-substituted BODIPY fluorescent turn-off probe for fluorescence imaging of hydrogen sulfide in living cells Deyan Gong a, Xiangtao Zhu b, Yuejun Tian c, Shi-Chong Han b, Min Deng a, Anam Iqbal a, Weisheng Liu a, Wenwu Qin a*, Huichen Guo b* a

Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu

Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China b

State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of

Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu, 730046, The People’s Republic of China c

Institute of Urology, the Second Hospital of Lanzhou University, Lanzhou, Gansu Pro vince, China

Corresponding author: Fax: +86-931-8912582 E-mail address: [email protected] (W. Qin); [email protected] (H. Guo) *

1

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ABSTRACT: Herein, A phenyl-selenium-substituted BODIPY (1) fluorescent turn-off sensor was developed for the purpose to achieve excellent selectivity and sensitivity for H2S detection based on the substitution reaction of phenylselenide group at the 3-position with H2S, the excess addition of hydrogen sulfide promoted the further substitution reaction at the phenylselenide group at the 5-position of the probe and was accompanied by a further decrease in fluorescence emission intensity. Sensor 1 demonstrated remarkable performance with 49-fold red colour fluorescence intensity decrease at longer excitation wavelength, a low detection limit (0.0025 µM) and specific fluorescent reply toward H2S over anions, biothiols as well as other amino acids in neutral media. It showed no obvious cell toxicity and good membrane permeability, which was exploited to intracellular H2S detection and imaging nicely through fluorescence microscopy imaging. Keywords: BODIPY; Selenium; Fluorescent probes; Hydrogen sulfide; Bioimaging

Introduction Hydrogen sulfide (H2S) is a weak acid in aqueous solution, equilibrating mainly with HS−, notorious for its unique odour of rotten eggs has emerged as a member of the endogenous gaseous transmitter of signalling molecules existing in the human body, which regulates cardiovascular, neuronal and immune systems.1-3 Research has also indicated that the abnormal level of H2S is related to diseases such as Down syndrome,4 arterial, diabetes, pulmonary hypertension and Alzheimer’s disease.5,6 The biological concentration of sulfide level range in 10–100 µM in plasma blood.7 2


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Hitherto, despite the fact that H2S has been linked to diverse physiological and pathological processes, large part of its underlying molecular events are still indistinct and could be deeply researched.5 Obviously, it is highly valuable to develop superior techniques or prospective chemical with remarkable selectivity and sensitivity to H2S detection in complicated biological systems. Coincident with the increased biological importance of H2S, new approaches for H2S quantification are rapidly emerging. Current strategies that are available for the detection of H2S include colorimetry,8 electrochemical assay,9 gas chromatography,10 and sulfide precipitation.11 However, these techniques do not allow the temporal and spatial monitoring of reactive and transient H2S and complicated sample preparation and tissues or cells destruction are required.12,13 Fluorescent imaging methods are highly desirable and offer high sensitivity as well as real-time imaging, a variety of innovative H2S fluorescent probes based on different reaction mechanisms have been reported, which rely mainly on reduction of azide and/or nitroso to amine mediated by H2S,14-16 nucleophilic addition of H2S,17-19 copper precipitation,20,21 thiolysis of leaving groups.12,22-24 Several fluorescent sensors containing selenium as leaving groups for detection of sulfhydryl group contained biomolecules have been explored.25-27 Seungyoon et al. has synthesised a phenyl-selenium-substituted coumarin sensor to detect glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) with excellent selectivity and fast reaction rate, which is based on the substitution reaction of phenylselenide group with sulfhydryl group of GSH.27 To our knowledge, 3



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reports about fluorescent probes containing phenylselenide group as the leaving groups to detect H2S are scarce. Various BODIPY derivatives as fluorescent probe with excellent performance have been synthesized for detection of varieties of sensing objects, like pH,28 cations,29

biothiols

and

so

on.30-32

3,5-diphenylselanyl

-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (1) as a fluorescence turn-off H2S sensor has been synthesized in this study according to the method of Seungyoon et al.27 Scheme 1 illustrates the feasible mechanism for its excellent sensitivity to H2S, which is based on the substitution reaction at phenylselenide group of 3-position with H2S, the excess addition of hydrogen sulfide promoted the further substitution reaction at the 5-position of the probe, realizing excellently sensitive and selective H2S detection for H2S triggered fluorescence turn-off nature.22,31,33 The excellent H2S sensing performance of 1 as well as its biological fluorescent labelling has been nicely visualized. c

d

Strong fluorescence

Weak fluorescence

Weaker fluorescence

4


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Scheme 1. Chemical structure of fluorescent probe BOD-PhSe (1) and a plausible mechanism of its reaction with H2S.

Experimental Instrument and reagent. Benzeneselenol (90%) was procured from Heowns. Sodium sulfide nonahydrate was purchased from Guangfu and was used as the hydrogen sulfide resource in all the experiments. (Caution! Benzeneselenol and H2S have a great unpleasant smell and are poisonous at higher concentrations. They should be handled with great care and in small amounts.) All experiments were carried out from commercially available reagents and solvents, and used without further purification, deionized water was used. NMR spectres were acquired on a Bruker spectrometer and mass spectra were taken on Bruker DRX-400 and Bruker microTOF-Q II mass spectrometer. Fluorescence spectra were recorded in FLS920 fluorescence spectrometer and Hitachi F-7000 spectrofluorimeter. Cresyl violet in methanol (λex = 582 nm, φf = 0.54) was used as fluorescence standard for quantum yield test.34 The

synthesis

of

probe

BOD-PhSe

(1).

As

shown

in

Scheme

1,

3,5-dichloro-BODIPY (2) was prepared according to the known procedure.35 1H NMR (CDCl3): δ 6.42 (d, 2H, J = 4.0 Hz, H-a), 6.83 (d, 2H, J = 4.0 Hz, H-b), 7.51 (m, 4H (ph)), 7.58 (t, 1H, J = 7.6 Hz, H-c); mass spectrum (ESI), m/z 336.9 (M); 316.9 (M-HF, 100%) ( C15H9BCl2F2N2 requires m/z 286.0715). 5



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Synthesis of BOD-PhSe (1): 100 mg (0.297 mmol) of compound 2 was dissolved in 25 ml acetonitrile，then 150 mg of anhydrous potassium carbonate (1.085 mmol) and 88 µL of benzeneselenol (90%, 0.742 mmol) were added successively; then the reaction mixture was heated under reflux. The reaction was completed after about 10 h (monitored by silica gel TLC using 5:1 petroleum ether–ethyl acetate). The organic layer concentrated under diminished pressure and then refined by silica gel flash chromatography (petroleum ether/ethyl acetate = 10:1) to give compound 1 as a deep purple solid (106 mg) in 62% yield, mp 107-109 oC. Rf 0.34 (petroleum ether/ethyl acetate = 5:1). 1H NMR (CDCl3): δ 5.892 (d, 2H, J = 4.4 Hz, H-a), 6.561 (d, 2H, J = 4.4 Hz, H-b), 7.430 (m, 11H (ph)), 7.753 (d, 4H, J = 6.4 Hz, H-d); 13C NMR (CDCl3): δ 120.73, 126.71, 127.81, 128.38, 129.10, 129.27, 129.57, 129.81, 129.98, 130.29, 131.59, 133.77, 136.55, 137.29, 138.37, 153.41. HRMS: calcd. for [1] (C27H19BF2N2Se2) 579.9940, found 579.9940. Measurements of photophysical properties and time-resolved emission spectra experiments. Surfactant Triton X-100 was used to improve the water-solubility of probe, 1 was diluted in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4) and then incubated at 37 oC in a thermostatic waterbath for the titration and selectivity experiment. Time resolved emission spectra experiments (TRES) were recorded using a FLS920 fluorescence spectrometer. The recorded column diagrams were fitted by Gaussian-weighted nonlinear least squares in the software package and the accurate 6


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results were obtained after changing parameters to get the minimum χ2. Global analyses were calculated through FAST software. Cell culture, confocal microscopy imaging. Baby hamster kidney (BHK) cells were incubated in culture medium overnight at 37 °C under a 5% CO2 and 95% relative humidity atmosphere. Cells were plated on glass coverslips, allowed to adhere for 24 h, and left incubating at 37 °C. Cells lines were treated with 1 (10 µM) in 1.0 mL of culture medium subjoined with 5% DMSO for 2 h; then, Na2S solution was added into the cells and incubated for another 30 min and washed three times with pre-warmed phosphate-buffered saline before imaging by Olympus FV1000-IX81 laser confocal microscope with an excitation wavelength of 559 nm. The other group was used as the control group, which was incubated merely with 1 (10 µM) under identical conditions for 2.5 h. Cytotoxicity tests. The in vitro cytotoxicity was tested using a standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay in cell lines. After BHK cells were incubated in culture media with 96-well plate overnight, different concentration of probe 1 was added to the wells of the treatment group and incubated for another 24 h. German Berthold Mithras2LB943 reader was used to measure the absorbance value at 450 nm.

Results and discussion Spectroscopic studies of BOD-PhSe as a Na2S fluorescent probe. The absorption and emission spectrums of 1 were recorded are shown in Figure S1. Ιn UV−vis 7



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absorption spectra, 1 exhibited a narrow band centred at 587 nm belongs to the 0−0 vibrational band of S0-S1 transition and a less pronounced shoulder (0−1 vibrational band of S0-S1 transition) at shorter wavelengths, which resemble those form previously reported BODIPY dyes. Moreover, the weaker, broader absorption band centred at ~390 nm belongs to the S0-S2 transition. 1 showed a fluorescence emission band centred at ~610 nm and φf value was 0.299. Spectroscopic/photophysical data of 1 is summarized in Table S1. Compare with phenoxy (phenyl-O-, λabs = ~520 nm and λem = ~530 nm,)36 and sulfur (-S-,λabs = ~570 nm and λem = ~580 nm,)37 disubstitution BODIPY analogue, not only the absorption and emission maxima of the phenyl-selenium-substituted BODIPY became deeper red. The properties of BODIPY dye show interesting optical changes when substitution groups change from oxygen to sulfur to selenium.38 Incubation of 100 µM Na2S with 1 (10 µM) in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4) at 37 oC after 20 min resulted a significant ~71 nm ( from 587 nm to 516 nm) blue shift in absorption spectrum and was accompanied with significant fluorescence intensity decrease (Figure S1, Table S1). The UV−vis absorption and fluorescence spectral changes of 1 with the reaction of Na2S are shown in Figure 1. With the increasing Na2S concentration in the range of 0−20 µM, intensity of the maximum absorption wavelength band centred at 587 nm of 1 increased with slightly hypsochromic shift, while new signals appeared at ~390 nm and ~516 nm with increasing intensity. The related peaks change at the 587/390 nm 8


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with increasing Na2S attribute to the substitution reaction with H2S at phenylselenide group at the 3-position of 1. In the Na2S concentration range of 20−85 µM, an excess addition of hydrogen sulfide promoted the further substitution reaction at phenylselenide group at the 5-position of the probe and was accompanied with the decrease of intensity of absorption band at around 516 and 552 nm, the absorption band at 587 nm was disappeared (Figure S1). A ratiometric absorption change with an isosbetic point at 540 nm was readily detected in the Na2S concentration range of 20−85 µM, indicates the presence of different species and the course demonstrated color variation from purple to pink in the sunlight

(Figure 1a, inset). Probe 1

exhibited a strong and broad emission band centered at 610 nm when excited at 582 nm. The increasing concentration of Na2S quenched the fluorescence emission intensity of 1 with a slightly hypsochromic shift from 610 nm to 602 nm. The quantum yield φf of 1 decreased from 0.299 in the absence of Na2S to 0.008 with addition of 10 equiv. of Na2S. Upon reacting with H2S, the phenylselenide group in 1 was substituted by the sulfhydryl group of H2S causing a 37-fold decrease in quantum yield and demonstrated a H2S-triggered fluorescence turn-off nature. As shown in Figure S 1c, when excited at 500 nm, probe 1 exhibited two weak broad emission bands centered at 558 nm and 609 nm, the weaker emission centered at 558 nm is assigned to the 0−1 vibrational band of the S0-S1 transition. Upon addition of different concentration of Na2S within 20 min, the fluorescence intensity of 1 at 609 nm (red) underwent a significant decrease while the broad emission at 558 nm (green) 9



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increased gradually. A distinct ratiometric fluorescence change with an isoemissive point at 588 nm was readily detected as the concentration of Na2S increased. The changes in fluorescence wavelengths also illustrate that the chemical transformation and the increasing emission at 558 nm attribute to the formation of sulfhydryl derivatives. The quantum yield of emission band at 558 nm was very low and the emission increases slightly at 558 nm after the addition of Na2S; as shown in the picture (Figure 1b, inset), there was almost no green fluorescence under a 365 nm UV lamp. It is not novel enough to apply 1 as a ratiometric fluorescence probe in consideration of the low sensitive detection of H2S; thus, further work was not carried out. The time-dependent fluorescence response of 1 (10 µM) in the presence of Na2S was investigated. Upon addition of 10 equiv. of Na2S, the emission band centred at 610 nm quickly decreased within 10 min and then slowly decreased and reached relative minimum about 20 min (Figure S2). Therefore, the detection of Na2S was achieved through the detection of probe 1 within 20 min. Figure 1c shows a plot of 1 quenched by Na2S and the correction curve of fluorescent strength (F) versus Na2S amount. F = 3472.09 −223.14 × [Na2S] (r = 0.9895, [Na2S] = 0−15 µM)

(1)

The emission band centered at 610 nm quickly decreased in correspondence with the amount of Na2S in the range of 0−15 µM and slowly decreased upon increasing the Na2S concentration in the range of 15−100 µM, the relative contributions of this 10


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trend are attributed in the same way as absorption signals changes in the two different ranges of Na2S concentration. The detection limit (DL) of 1 for Na2S was calculated to be 0.0025 µM (0−15 µM) from the calibration curve with the equation: DL = 3.3σ/k (σ: the standard deviation of fluorescence spectrometer, k: the slope of the correction curve). As shown in Table S2, the DL acquired by our strategy was well below than the reported ranges. Comparative tests have been conducted to examine the effect of the usage of Triton X-100 and DMSO on the fluorescent detection experiments. When no triton was added, the usage of DMSO should be increase to dissolve the probe 1 well; DMSO/HEPES buffer (1: 1, v/v) was a suitable proportion for our test. Figure S3 shows time-dependent fluorescence spectra, titration and selective fluorescence spectra of probe 1 for Na2S. The reaction of Na2S in DMSO/HEPES buffer (1: 1, v/v) showed similar fluorescence response upon addition of Na2S and biothiols as in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v) (Figure S3b, c). On the one hand, DMSO of up to 50% volume ratio had to be used together with the probe. High percentage of organic solvent is probably harmful for the cells and limits its range of uses. On the other hand, as is shown in the time course in Figure S3a, upon addition of 10 equiv. of Na2S, the emission band centred at 610 nm decreased slowly and reached relative minimum about 50 min. Therefore, the reaction rate of Na2S in DMSO/HEPES buffer (1: 1, v/v) was slower than in Triton X-100/DMSO/HEPES

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buffer (0.01: 1: 9, v/v/v). Thus, Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v) was a better choice for the detection of H2S.

12


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Figure 1. (a) Absorption and (b) Fluorescence changes of 1 (10 µM) with Na2S. Inset: a. visible and b. fluorescence change under a 365 nm UV lamp of 1 with Na2S. (c) The calibration curve of 1 quenched by Na2S excited at 582 nm. Relative standard deviation (RSD) is 7.5%，n = 4. All spectra were obtained after 20 min of incubation of 1 with100 µM Na2S.

Mechanism exploration. According to the literatures reported, upon reacting with H2S, group of fluorophore substituted by the sulfhydryl group of H2S would quench the fluorescence for H2S triggered fluorescence turn-off nature. Scheme 1 illustrates the feasible mechanism for its excellent sensitivity to H2S. To identify the substitution reaction with H2S, the 1H NMR spectra of 1 in a solution of DMSO-d6 with Na2S• 9H2O (1 equiv. or 7 equiv.) at 30 min reaction time was monitored at 25 °C (Figure 2). Because of the symmetrical structure of 1, the signals of the pyrrole rings’ four 13



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protons of 1 appear at 5.86, 6.70 ppm, respectively. After 30 min of incubation with 1 equiv. Na2S•9H2O, the 1H NMR spectral peaks belong to the phenylselenide group at 7.75 ppm transferred to the multiple peaks range in 7.16−7.30 ppm for the leaving of phenylselenide group. The 1H NMR peaks belong to the pyrrole rings’ protons at 5.86 ppm transferred to 5.98 ppm and two new peaks at 6.03 and 6.10 ppm assigned to the pyrrole rings emerged concomitantly. It is confirmed that the symmetrical structure of 1 was destroyed by the substitution reaction with H2S at phenylselenide group at the 3-position. MS Spectrum of 1 (Figure S4) in EtOH with Na2S (1 equiv.) at 2 h reaction time at 25 °C was measured to characterize this reaction further. The predicted product is BOD-SH (the molecular weight of [BOD-SH] (C21H15BF2N2SSe) is 456.0). In Figure S4, m/z 495.1 (calcd = 495.0) belongs to [BOD-SH +K] was clearly observed. The 1H NMR peaks belong to the pyrrole rings all transferred to the range of 6.52−6.60 ppm and formed multiple peaks with 7 equiv. of excess Na2S (Figure 2). The multiple peaks range in 6.52−6.60 ppm assigned to the symmetrical pyrrole rings’ protons of BOD-2SH because of the further substitution reaction at phenylselenide group at the 5-position of 1. After treatment with Na2S, the two phenylselenide groups of 1 were substituted by sulfhydryl group in a two-step substitution reaction.

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e

d

b

a

e'

d'

b'

a'

e''

d''

b''

a''

c

a)

c

b,d

b)

a,e

b'

d' e' a'

a'',b'',d'',e''

c) 8 .0

7 .8

7 .6

7 .4

7 .2

7 .0

6 .8

6 .6

6 .4

6 .2

6 .0

5 .8 p p m

Figure 2. Partial 1H NMR spectra in DMSO-d6 at 25 °C: (a) Probe 1; (b) Probe 1 with

Na2S•9H2O (1 equiv.), 30 min; (c) Probe 1 with Na2S•9H2O (7 equiv.), 30 min.

Selective fluorescence response of 1 turn-off fluorescence towards Na2S. To

investigate the selectivity in aqueous solution, various representative intracellular species were added into the solution of chemodosimeter 1 (10 µM) in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4). As shown in Figure 3, only Na2S decrease the fluorescence intensity dramatically with about 49-fold, while other analytes were silent to 1 under the same condition. Figure S5a shows the detailed emission response spectra of 1 upon addition of 10 equiv. of biothiols and Na2S and Figure S5b shows the competition graph of 1 in the presence of 10 equiv. of biothiols with Na2S. It clearly shows that the turn-off fluorescence response of 1 with

15



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Na2S was also demonstrated significantly and was scarcely disturbed by concomitant biothiols. In addition, 1 showed pH-dependence in the detection of H2S. As shown in Figure S6, the fluorescence intensity of 1 was slightly changed in the pH value scope of 5−9. Moreover, in the incubation of Na2S, the turn-off fluorescent response also demonstrated significantly and maintains relatively steady in wide pH scope of 6−9; however, the exception displayed at pH 5 implied the thiolysis reaction of phenylselenide group could not proceed to a great extent with Na2S in acid surrounding. 1 can be used for the detection of H2S at a wider pH range (pH 6-9), within which the majority of biological samples (5.25−8.93) can be detected. pH value alterations show no strong disturbance to H2S detection of chemodosimeter 1 in solution. To examine the reversibility of the processes, an amount of benzeneselenol was added to the 1–Na2S mixture solution (1 (10 µM) reacted with 40 µM Na2S in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4) at 37 oC for 20 min was prepared as the 1–Na2S mixture solution). The fluorescence spectra of 1–Na2S as a function of benzeneselenol concentration are shown in Figure S7. Upon increasing benzeneselenol concentration, the emission intensity with a maximum at 610 nm increased slightly in the range of 0−180 µM and then remained constant. It is not novel enough to apply 1 as fluorescence probe for the detection of benzeneselenol in

16


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consideration of the low substitution reaction rate, probability and sensitivity with benzeneselenol; thus, further work was not carried out. 50

Fluorescence Decrease (F0/F)

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40

30

20

10

0 1 2 3 4 5 6 7 8 9 1011 12 1314 15 1617 181920 21 2223 24 25262728 2930 31 3233

Figure 3. Fluorescence responses at λem = 610 nm of 1 (10 µM) reacted with 0.1 mM

different kinds of species. All spectra were obtained after 50 min of incubation with different analytes, RSD is about 6.4%，n = 4, λex = 582 nm. 1) Probe 1, 2) Na2S, 3) HSO3−, 4) F−, 5) Cl−, 6) Br−, 7) I−, 8) CH3COO−, 9) SCN−, 10) N3−,11) NO3−, 12) SO42−, 13) CO3−, 14) H2PO4−, 15) ClO−, 16) H2O2,17) Pro, 18) Ile, 19) Ala, 20) His, 21) Glu, 22) Tyr, 23) Lys, 24) Met, 25) Asp, 26) Phe, 27) Arg, 28) Gly, 29) Ser, 30) Cys, 31) Hcy, 32) GSH, 33) ascorbic acid.

The fluorescence decay traces of 1 toward different concentrations of Na2S.

Fluorescence decay traces of 1 or 1 at different concentrations of Na2S in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4) at 37 oC were tracked at several emission wavelengths through a single-photon timing method.39 For 17



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1–Na2S mixture solution, global analyses in terms of decay time τi and αi were

performed incorporating in a single decay surface curves recorded 20 min after Na2S addition at the same excitation wavelength (560 nm), but at different emission wavelength (590, 610, 630 and 650 nm) (Table S3, Figure S8, Figure S9). τi were linked for decay traces measured at different λem by using FAST software. It can be expected global analysis with 4 decay traces would estimate the {τi, αi} values with higher accuracy than single-curve analysis. The fluorescence decay spectra of probe were recorded and globally analyzed as bi-exponential profiles. With the increasing detection wavelength λem in the range of 590-650 nm, there displayed an enhancement in the related amplitude of the slow constituent τ1 (α1 = 1% at 590nm, α1 = 4% at 610nm, α1 = 5% at 630nm, α1 = 5% at 650nm) (Table S3). τ2 = ~1.04 ns as the major decay constituent belong to the fluorescence lifetime of BODIPY chromophore, while τ1 = ~2.73 ns could be attributed to the lifetime of formation of aggregates (formed because of the π, π-stacking effect.) of 1 in Triton X-100/DMSO/HEPES buffer. Upon addition of different concentration of Na2S to 1, the fluorescence decay changed obviously in the presence of Na2S with the amount of 0, 10, 25, and 100 µM after 20 min (Figure 4). Triexponential analyses were applied incorporating in decay spectra; detailed photophysical properties of sensor 1 about decay times τ1, τ2 and τ3 were listed in Table S3. At the detection emission wavelength of 610 nm, τ1 and τ2 of the mixture solution were almost constant (~2.73 and ~1.04 ns), but the longer lifetime increased with the amplitude (4% to 40%) and the shorter lifetime decreased with the amplitude (96% to 43%). There was a new decay time τ3 (~0.49 ns) appeared and then the amplitude (20% to 17%) remained almost constant upon the increasing concentration of Na2S. The new fast decay can explain as in the presence of Na2S, the intramolecular charge transfer (ICT) from the negatively charge of -S− to the 18


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electron-withdrawing BODIPY moiety was enhanced. In general, the decay profiles of 1 in addition of Na2S are complicated due to the involvement of at least two or three different species and the likely presence of multiple states. A possible explanation for the absorption, fluorescence emission, and lifetime changes of 1 with Na2S is the mono- or disubstituted process of -SH indicates the presence of three different species. One is disubstituted-phenyl-selenium 1 (λabs = 587 nm and λem = 610 nm) with strong fluorescent, the other is mono-substituted -phenyl-selenium BOD-SH (λabs = 558 nm) with low fluorescent. The last one is BOD-2SH (λabs = 500 nm) with low fluorescent. It was proposed the acid-base

equilibrium of BOD-SH ⇋ BOD-S− has happened at pH = 7.4. The φf value of monoor disubstitution of 3,5-dichloro-BODIPY chromophore with S-nucleophile group are quite high.37 Because of the excited-state ICT effect from the negatively charged -S– (BOD-SH ⇋ BOD-S−) to BODIPY acceptor which is electron deficient, φf value of the sulfhydryl derivatives product were low. .

Figure 4. Representative time-resolved emission spectra of 1 (10 µM) in the absence

and presence of different concentration of Na2S in Triton X-100/DMSO/HEPES buffer (0.01: 1: 9, v/v/v, 10 mM, pH 7.4) and 1, all spectra were obtained after 20 min of incubation with Na2S, λex = 560 nm, λem = 610 nm. 19



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Visualizing Na2S in living cells. As mentioned above, 1 showed little fluorescence

response to other anions and biological thiols, which made the 1 promising for specific recognition of aqueous sulfide in live cells. Initially, the BHK cells were treated with 1 to assess the capability and its potential in bioimaging of H2S. Prior to the bioimaging studies, a good level of cell viability, low cytotoxicity and good biocompatibility in the presence of 1 was confirmed by MTT assay (Figure S10). As a control, fluorescent confocal images of BHK cells treated with and without Na2S were both taken and compared. It clearly indicated that the 1-stained cells not only kept good shape and appeared viable but also displayed remarkable red emission attributed to the fluorescence of 1 under the monoemission mode imaging (Figure 5a). Overlays of confocal fluorescence and bright-field images demonstrated that the fluorescence was evident in the cytoplasm over the nucleus and the membrane. The other group treated with Na2S displayed negligible fluorescence when 1-treated cells were then exposed to 100 µM Na2S for 30 min at 37 °C (Figure 5b). Clearly, these data demonstrate the potential of 1 as a sensitive fluorescent probe for specific recognition of aqueous sulfides and fluorescent image analysis can be successfully used to monitor the presence of Na2S.

20


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Bright-field

Red channel

Overlay

(a)

(b)

Figure 5. BHK cells confocal fluorescence microscopy imaging: (a) Fluorescence

images of BHK cells incubated with 1 (10 µM). (b) Images of BHK cells incubated with 100 µM Na2S and 1 (10 µM). Scale bar = 7.5 µm.

Conclusions In conclusion, a novel fluorescent turn-off sensor 1 for H2S based on the two steps substitution reaction with the H2S at phenylselenide group of 1 was identified, which is very helpful for the further design of excellent fluorescent probes for H2S detection. Sensor 1 displayed an excellent selective sensitivity fluorescent respond towards H2S, which afforded a DL of 0.0025 µM (0-15 µM). More notably, the chemodosimeter 1 could be demonstrated to label cells and monitor H2S in living cells by confocal fluorescence imaging successfully. The design strategy and H2S triggered fluorescence turn-off nature reported here should be applicable to develop a wide range of practically H2S fluorescent sensors with high selectivity and sensitivity. 21



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Acknowledgments

We acknowledge the Natural Science Foundation of China (No. 21271094), the Ministry of Science and Technology of China (2014DFA31890) and the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant no. J1103307). Supporting information

Please

see

supporting

information

(SI)

for

the

details

of

Spectroscopic/photophysical data and figures (Figure S1-S10). The material of supplementary data and compound characterization data also can be found. The Supporting Information is available free of charge on the ACS Publications website. References

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“for TOC only”

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A Phenylselenium-Substituted BODIPY Fluorescent Turn-off Probe for

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