A novel fluorescent probe for rapid and sensitive detection of hydrogen sulfide in living cells

    A novel fluorescent probe for rapid and sensitive detection of hydrogen sulfide in living cells Jian Pan, Junchao Xu, Youlai Zhang, Liang Wang, Caiqin Qin, Lintao Zeng, Yue Zhang PII: DOI: Reference:

S1386-1425(16)30312-2 doi: 10.1016/j.saa.2016.05.054 SAA 14475

To appear in: Received date: Revised date: Accepted date:

28 May 2015 24 May 2016 31 May 2016

Please cite this article as: Jian Pan, Junchao Xu, Youlai Zhang, Liang Wang, Caiqin Qin, Lintao Zeng, Yue Zhang, A novel fluorescent probe for rapid and sensitive detection of hydrogen sulfide in living cells, (2016), doi: 10.1016/j.saa.2016.05.054

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ACCEPTED MANUSCRIPT

A novel fluorescent probe for rapid and sensitive detection

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of hydrogen sulfide in living cells

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Jian Pan a, b, Junchao Xu a, b, Youlai Zhang b, Liang Wang b, Caiqin Qin a, Lintao Zeng a, *, Yue Zhang b,*

a

Department of Chemistry and Material Sciences, Hubei Engineering University,

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b,

Hubei Xiaogan 432000, P. R. China. E-mail: [email protected] (L. Zeng); School of Chemistry & Chemical Engineering, Tianjin University of Technology,

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b

Tianjin 300384, P. R. China. Fax: (+86) 22 60214252; E-mail: [email protected]

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(Y. Zhang).

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ABSTRACT

indole-BODIPY,

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A novel fluorescent probe for H2S was developed based on a far-red emitting which

was

decorated

with

morpholine

and

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2,4-dinitrobenzenesulfonyl (DNBS) group. This probe showed rapid response (t1/2 = 3 min), high selectivity and sensitivity for H2S with significant colorimetric and fluorescence

OFF-ON

signals,

which

was

triggered

by

cleavage

of

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2,4-dinitrobenzenesulfonyl group. This probe could quantitatively detect the concentrations of H2S ranging from 0 to 60 µM, and the detection of limit was found to be as low as 26 nM. Cell imaging results indicated that the probe could detect and visualize H2S in the living cells. Keywords: Fluorescent probe;

BODIPY;

Hydrogen sulfide

Imaging

ACCEPTED MANUSCRIPT 1. Introduction Hydrogen sulfide (H2S) is an important gaseous transmitter, which is endogenously

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generated from cystein with the aid of cystathionine β-synthase [1], cystathionine

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γ-lyase [2], and 3-mercaptopyruvate sulphur transferase [3]. H2S plays vital roles in

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various physiological processes, such as modulation of blood pressure [4], reduction of ischemia reperfusion injury [5, 6], exertion of anti-inflammatory effects [7] and reduction of metabolic rate [8]. However, aberrant H2S production is associated with

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pathological states including Alzheimer's disease [9], Huntington’s disease [10], and Parkinson’s disease [11]. Thus, visualization of the production and concentration of

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H2S within living cells is beneficial to the early diagnose of these diseases. Fluorescence-based probe is a powerful tool for the detection and visualization of some biological species

[12 — 18] due to its non-invasiveness, high sensitivity,

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high temporal and spatial resolution [19, 20]. Recently, a number of fluorescent

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probes for H2S have been developed on the basis of H2S-mediated reduction of azides

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[21 — 23] and nitros [24, 25], nucleophilic addition reaction [26, 27], Tandem Michael addition reaction [28, 29], copper sulfide precipitation [30, 31], and thiolysis of dinitrophenyl ether [32, 33]. Xian’s group has utilized disulfide exchange

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mechanism to devise some fluorescent probes for H2S [34 — 36]. In addition, enzyme-based probe have also been proposed [37]. Although these probes are innovative, they still have some disadvantages, such as excitation/emission in the ultraviolet or visible region [28, 33], and long response time (up to 20 min). Excitation/emission in the visible region might be subjected to the interference from background [38, 39]. Long response time is not suitable for real time analysis and bio-imaging of H2S due to its transient nature. Therefore, it is highly desired to develop some fluorescent probes with long emission wavelength, rapid response and high sensitivity. Herein we report a new fluorescent probe for H2S based on indole-BODIPY fluorophore (Scheme 1). This probe exhibited rapid response to H2S with good selectivity, long emission wavelength (λem = 635 nm) and low detection limit.

ACCEPTED MANUSCRIPT Moreover, the probe has been successfully used for imaging H2S in living cells with satisfying results.

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2. Experimental section

2,4-Dimethylpyrrole,

trifluoroacetic

1,4-benzoquinone

(DDQ),

acid

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2.1. Materials (TFA),

2,3-dichloro-5,6-dicyano-

1H-indole-3-carbaldehyde, chloride,

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2,4-dinitrobenzenesulfonyl

piperidine,

4-(2-chloroethyl)morpholine,

4-hydroxybenzaldehyde and triethylamine were purchased from commercial suppliers

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(Aladdin-Reagent, Sigma-Aldrich, TCI), and used without further purifications. 4,4-difluoro-8and

1-(2-morpholinoethyl)-1H-indole-3-carbaldehyde

were

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za-s-indacene

(4-hydroxyphenyl)-1,3,5,7–tetramethyl-4-bora-3a,4a-dia-

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synthesized according to literatures [39, 40].

The

1

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2.2. Equipments and methods H NMR and

13

C NMR spectra were recorded on a Bruker AV-400

spectrometer with tetramethylsilane (TMS) as the internal standard. The chemical

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shift was recorded in ppm and the following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet. Mass spectra were measured by a HP-1100 LC-MS spectrometer. UV-vis spectra were recorded on a Hitachi UV 3310 spectrometer. Fluorescence spectra were recorded on a Hitachi FL-4700 fluorometer. Fluorescent images were acquired on a Nikon A1 confocal laser-scanning microscope with a 100x objective lens. Solvents used for UV-vis and fluorescence measurements were of HPLC grade. Column chromatography was performed on silica gel (mesh 200–300), which was purchased from Qingdao Ocean Chemicals Corporation.

ACCEPTED MANUSCRIPT 2.3. Synthesis and characterization OH OH

Piperidin, AcOH

N +

N

N B F F

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N

N B F F

N

Yield: 82% O 2 NO2 O S O Cl

O2 N O2 N

O SO O

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Et3N, CH2Cl2

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1 O2 N

Toluene, reflux

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O

N O

3

NaHS

N

O

N

DNBY

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Yield: 90%

N N B F F

N

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Scheme 1 Synthetic scheme of the probe DNBY. 2.3.1. Synthesis of compound 3

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4,4-difluoro-8-(4-hydroxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (170.0 mg, 0.50 mmol), 1-(2-morpholinoethyl)-1H-indole-3-carbaldehyde (168.0 mg, 0.65 mmol) and two drops of piperidine were dissolved in 30 mL of toluene. The reaction mixture was heated to 110 °C under nitrogen atmosphere, then two drops of acetic acid were added. The reaction mixture was stirred at 110 oC for 16 h. After the reaction was finished, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2/C2H5OH = 60/1) to afford compound 3 as a dark blue solid (179.9 mg, 0.31 mmol, 62%). 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.93 (s, 2H), 7.79 (d, J = 16.4 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 16.4 Hz, 1H), 7.28 (m, 2H), 7.15 (d, J = 8.2 Hz, 2H), 6.99 (s, 1H), 6.93 (d, J = 8.2 Hz, 2H), 6.10 (s, 1H), 4.38 (t, J = 5.7 Hz, 2H), 3.56 (s, 4H), 2.70 (s, 2H), 2.48 (s, 6H), 1.50 (s, 3H), 1.42 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 158.47,

ACCEPTED MANUSCRIPT 155.61, 151.25, 143.37, 139.94, 139.55, 137.75, 133.39, 132.80, 131.22, 129.87, 125.95, 125.19, 123.12, 121.52, 120.48, 120.18, 118.27, 116.39, 113.78, 113.59, 111.46, 66.64, 57.99, 53.73, 43.49, 15.01, 14.73, 14.47. HR-MS (ESI): calcd for

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C34H35BF2N4O2+H 581.2905; Found 581.2906

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2.3.2. Synthesis of compound DNBY

Compound 3 (81.0 mg, 0.14 mmol) and triethylamine (60.0 μL, 0.35 mmol) were in

20

mL

of

anhydrous

CH2Cl2.

To

this

solution,

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dissolved

2,4-dinitrobenzenesulfonyl chloride (74 mg, 0.28 mmol) in 2 mL of anhydrous

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CH2Cl2 was added dropwise in 0.5 h at 0 °C. Then, the temperature was raised to 40 °C, and the reaction mixture was stirred for 2 h. After the reaction completed, the solvent was removed under reduced pressure. The residue was purified by silica gel

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column chromatography (CH2Cl2/C2H5OH = 100/1) to afford the probe DNBY as a

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dark blue solid (70.0 mg, 0.09 mmol, 64%). 1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 2.0 Hz, 1H), 8.60 (dd, J = 8.7, 2.2Hz, 1H), 8.17 (d, J = 8.7 Hz, 1H), 7.96 (s, 1H),

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7.94 (d, J = 6.9 Hz, 1H), 7.84 (d, J = 15.1 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.56 – 7.34 (m, 6H), 7.28 (m, 2H), 7.05 (s, 1H), 6.12 (s, 1H), 4.38 (s, 2H), 3.55 (s, 4H), 2.70 (s, 2H), 2.48 (s, 6H), 1.35 (s, 3H), 1.28 (s, 3H).

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C NMR (100 MHz, DMSO-d6) δ

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156.42, 152.06, 151.64, 149.39, 148.67, 143.12, 139.43, 137.77, 136.54, 135.13, 134.34, 133.72, 132.79, 131.39, 130.57, 130.33, 127.70, 125.99, 123.33, 123.22, 121.62, 120.78, 120.18, 118.90, 113.87, 113.35, 111.52, 66.46, 57.78, 53.62, 43.30, 15.05, 14.72, 14.46. HR-MS (ESI): calcd for C40H37BF2N6O8S+H 811.2533; Found 811.2532. 2.3.3. Conversion of compound DNBY by NaHS NaHS (10.0 mg, 0.18 mmol) and compound DNBY (30.0 mg, 0.037 mmol) were dissolved in 3.0 mL absolute ethanol at room temperature. 30 min later, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (CH2Cl2/C2H5OH = 60/1) to give a dark blue solid (11.6 mg, 0.02

ACCEPTED MANUSCRIPT mmol, 54 %).

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2.4. Fluorescence analysis

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The solutions of various testing species were prepared from CaCl2, MgCl2, KI, NaCl, KBr, NaF, NaN3, Na2SO4, Na2SO3, CH3COONa, NaH2PO4, GSH, Cys, Hcy,

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H2O2 in double-distilled water. Hydroxyl radicals were generated from the reaction of Fe2+ with H2O2. Singlet oxygen (1O2) was generated from ClO－and H2O2. The stock

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solution of DNBY (10 µM) was prepared in 10 mM PBS buffer solution (pH 7.4) with 30% fraction of ethanol. For all measurements, the excitation wavelength was

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560 nm, the excitation and emission slit widths were 5 nm. 2.5. Determination of the detection limit

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The fluorescence spectrum of probe DNBY (10 µM) in the absence of HS－was

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measured for five times to obtain the standard deviation of a blank measurement. The fluorescence intensity at 635 nm was plotted as a concentration of HS－. The detection

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limit was calculated base on signal to noise ratio (S/N =3).

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2.6. Determination of the fluorescence quantum yield Rhodamine B (Φr = 0.65 in ethanol) was used as a reference to calculate the quantum yield of compound 3 in ethanol according to the following equation:

 f Fs Ar   r Fr As

 ns   nr

  

2

where, As and Ar were the absorbance of the sample and the reference, respectively. Fs and Fr are the corresponding integrated fluorescence intensities, and n is the refractive index of the solvent.

ACCEPTED MANUSCRIPT 2.7. Cell culture and fluorescence imaging HeLa cells (Perking Union Medical College, China) were cultured in Dulbecco’s

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modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum

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(Invitrogen Corp., Carlsbad, CA) and penicillin (100 units/mL)-streptomycin (100

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μg/mL) liquid (Invitrogen Corp., Carlsbad, CA) at 37°C in a humidified incubator containing 5% CO2 and 95% air. The cells were incubated for 2 days before dye loading on an uncoated 35 mm diameter glass-bottomed dish (D110100, Matsunami,

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Japan). Then, the cells were incubated with DMEM containing 10% FBS and 10 μM probe DNBY for 30 min at 37 °C, washed with PBS three times, and mounted on the

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microscope stage. Fluorescence images were captured using a Nikon A1 Application. The cells were furthermore incubated with NaHS for 15 min, and then washed with PBS twice for confocal laser-scanning microscopy measurement. Fluorescence

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images were captured using a Nikon A1 Application.

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3. Results and discussion

3.1. Design and Synthesis of probe DNBY

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BODIPY has been recognized as a versatile fluorescent dye because of its outstanding photophysical properties [41, 42], such as high quantum yield, good photostability and chemical stability. For bio-imaging, a fluorescent probe with high quantum yield and long emission wavelength is highly desired, because it can minimize the interference from the background [43, 44]. To push the absorption and emission wavelength into the far-red or near infrared region, BODIPY was condensed with indole-3-carbaldehyde to extend the π-conjugation. To construct a photo-induced electron transfer (PET)-based fluorescent probe for H2S, 2,4-dinitrobenzenesulfonyl (DNBS) group was employed, as shown in Scheme 1. At the molecular level, H2S acts as a good reducing agent and a good nucleophile. So, 2,4-dinitrobenzenesulfonyl group of the probe DNBY would be removed or reduced by HS-, resulting in a significant fluorescence enhancement.

ACCEPTED MANUSCRIPT The probe DNBY was synthesized in two steps, as shown in Scheme 1. BODIPY was condensed with 1-(2-morpholinoethyl)-1H-indole-3-carbaldehyde in the presence of piperidine and acetic acid, affording the compound 3 in 62% yield. Then,

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compound 3 was treated with 2,4-dinitrobenzenesulfonyl chloride to produce probe DNBY with large through-put (90%). The structures of DNBY and compound 3 were

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3.2. The optical response of probe DNBY to H2S

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fully characterized by 1H NMR, 13C NMR and HR-MS (ESI†).

The UV-vis absorption spectrum of probe DNBY was measured in PBS solution

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(C2H5OH/PBS = 3:7, pH=7.4), as shown in Fig. 1a. DNBY displayed a broad absorption band centered at 620 nm (Fig. 1a inset). Upon the addition of 50.0 equiv. of NaHS, the maximum absorption wavelength of the probe shifted to 590 nm, and

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the color of DNBY solution changed from blue to red. Notably, the UV-vis absorption

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spectrum of probe after treatment with 50.0 equiv. of NaHS was almost the same with that of compound 3 (Fig. 1a, black line), suggesting that the DNBS group was

eyes.

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removed by HS−. This distinct color change was useful for detection of HS− by naked

As shown in Fig. 1b, the free probe was non-fluorescent in PBS solution

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(C2H5OH/PBS = 3:7, pH=7.4) due to 2,4-dinitrobenzenesulfonyl group. Upon the addition of an increasing amount of NaHS, the fluorescence intensity at 635 nm increased progressively. When the concentration of NaHS was 50.0 equiv. with respect to the probe, the fluorescence intensity of the probe reached a plateau and a large fluorescence enhancement (~ 200 folds, Φ = 0.21 in PBS) was observed (Fig. 1b inset). The fluorescence OFF–ON switch might be triggered by cleavage of the 2,4-dinitrobenzenesulfonyl group [45]. It was noteworthy that a maximal fluorescence change was obtained within 10 min in the presence of 50 equiv. of NaHS (shown in Fig. 2a), which was impressive compared with some reported H2S probes (sometimes required > 20 min). Moreover, the probe showed a strong fluorescence band at 635 nm in the presence of NaHS, which was suitable for bio-imaging due to its far-red

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0.6 0.4 0.2 0.0 520 540 560 580 600 620 640 660 680 700

50 equiv.

6000 4000

0

2000

0 600

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Wavelength(nm)

(b)

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0.8

8000

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DNBY DNBY+NaHS Compound 3

(a)

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1.0

Fluorescence Intensity

Normalized Absorbance

region emission and bright fluorescence.

620

640

660

680

700

Wavelength(nm)

Fig. 1. (a) Absorption spectra of DNBY (10 μM) in the absence (blue line) or

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presence (red line) of NaHS (500 μM) in PBS aqueous solution (C2H5OH/PBS = 3:7, pH 7.4). Inset: color changed from blue to red. (b) Fluorescence spectra changes of

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DNBY (10 μM) upon the addition of NaHS (0 – 500 μM) in PBS aqueous solution

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(C2H5OH/PBS = 3:7, pH 7.4). Slits: 5/5 nm. We also explored the ability of probe DNBY to quantitatively detect HS− in PBS

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solution. DNBY (10 μM) was treated with different concentrations of NaHS (0 – 500 μM), and the fluorescence spectra were recorded. By plotting the fluorescence intensity at 635 nm versus the concentration of NaHS, a good calibration curve was

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obtained, as shown in Fig. 2b. The fluorescence intensity was linearly related to the concentration of HS− ranging from 0 to 60 µM, and the detection of limit was calculated to be as low as 26 nM base on signal to noise ratio (S/N =3). The fast response and excellent linear relationship provided a real-time quantitative detection method for HS−. Next, we used fetal bovine serum to investigate whether our probe could detect HS− in complex biological samples. Following the above method, we got another calibration curve between fluorescence intensity at 635 nm and the concentration of HS−. The regression equation was I635 = 34.41[HS−] + 234.2 (Fig. 2d) with R2 = 0.99. These results indicated that DNBY could quantitatively detect HS− in complex biological systems.

5000 4000 3000 2000 1000 0

200

400

600

8000 6000 4000

0

1500

0

1000 500

600

620

640

660

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0

680

3500

10

20

30

40

[HS-] (μM)

50

60

(d)

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2000

Fluorescence Intensity

40 equiv.

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Fluorescence Intensity

4000

(c)

y=162.56x+313.77 R2=0.9957

2000

0

800

Response Time (s) 2500

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6000

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7000

0

(b)

10000

(a)

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8000

Fluorescence Intensity

Fluorescence Intensity

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700

3000 2500 2000 1500

y=34.41x+234.2 R2=0.9915

1000 500 0 0

20

40

60

[HS-] (μM)

80

100

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Wavelength(nm)

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Fig. 2. (a) Time-dependent fluorescence intensity changes of DNBY (10 μM in C2H5OH/PBS = 3:7, pH 7.4) in the presence of 50.0 equiv. of NaHS. (b) The linear relationship between fluorescence intensity of DNBY at 635 nm and the concentration

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of NaHS. λex = 560 nm. Slits: 5/5 nm. (c) Fluorescence spectra titrations of DNBY (10 μM) with NaHS in the fetal bovine serum (C2H5OH/ fetal bovine serum = 3:7). λex = 560 nm. Slit: 2.5/2.5 nm. (d) The linear relationship between the fluorescence intensity at 635 nm and the concentration of NaHS in the fetal bovine serum (C2H5OH/ fetal bovine serum = 3:7). λex = 560 nm. Slits: 2.5/5 nm. 3.3. pH-dependent fluorescence response of the probe to H2S The pH-dependent fluorescence responses of the probe to HS− were also investigated, as shown in Fig. 3. The fluorescence intensity of the probe remained constant from pH 2.0 to 8.5, suggesting that this probe was very stable for a wide range of pH values. Upon the addition of 50.0 equiv. of NaHS, the fluorescence intensity of DNBY increased drastically. Whereas, pH has some influence on the

ACCEPTED MANUSCRIPT sensing property of DNBY, as shown in Fig. 3. In the presence of 50.0 equiv. of NaHS, the fluorescence intensity of DBNY was much weaker at high pH values, because the electron-rich morpholine unit quenched the fluorescence of DNBY

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through a photoinduced electron transfer (PET). In acidic environment, the morpholine was protonated, so the PET was inhibited and the fluorescence was

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recovered. According to the previous study [46, 47], morpholine would be protonated

DNBY DNBY+NaHS

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2500

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2000 1500

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1000 500 0

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Fluorescence Intensity

only in lysosomes (pH 4.5−5.5) on account of its pKa.

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2

3

4

5

6

7

8

9

pH

Fig. 3. Fluorescence intensity changes of DNBY (10 μM, C2H5OH/PBS = 3:7) in the absence (■) or presence (●) of NaHS (500 μM) at different pH values. The

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fluorescence intensity of DNBY upon treatment with NaHS was collected after 15 min at room temperature. λex = 560 nm, λem = 635 nm. Slits: 2.5/2.5 nm. 3.4. Selectivity of the probe DNBY toward H2S Selectivity is an important parameter for all kinds of detection methods. To evaluate the selectivity of the probe DNBY for HS−, various biologically relevant species were examined including some representative anions, metal ions, reactive oxygen species, small-molecule thiols, and NaHS. As shown in Fig. 4, the probe showed a negligible response to some representative anions (F−, Cl−, Br−, I−, SO42−, AcO−, N3−, H2PO4−), metal ions (K+, Na+, Mg2+, Ca2+ ), reactive oxygen species (H2O2, •OH, 1O2 ), and reducing agents (SO32−) at the biologically relevant concentrations. Besides, some

ACCEPTED MANUSCRIPT bio-thiols such as glutathione (GSH) and DL-homocysteine (DL-Hcy), only induced a very small fluorescence enhancement (< 15 folds). By contrast, a significant color change and a great fluorescence enhancement of the probe were obtained in the

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presence of 50.0 equiv. of NaHS, which could be observed by naked eyes (shown in Fig. 4b and c).

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Although the probe also displayed fluorescence response to L-cysteine (L-Cys), the fluorescence intensity was much weaker in contrast to that of the probe DNBY. HS− has much higher nucleophilic reactivity and the reaction kinetic constant was at least

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one order of magnitude larger than that of bio-thiols (shown in Fig. 5 and Fig. S1). These results suggested that the probe has good selectivity for HS− over other anions

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and biological species. Therefore, the probe has potential applications for the

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8000

Competing species+NaHS

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Competing species

7000

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6000 5000 4000 3000 2000

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(a)

Fluorescence Intensity

detection of HS− in complex biological environments.

1000

H 2O GS 2 H Hc y Cy s

O

·OH2

3 1

Bla

nk K+ Na + Mg 2+ Ca 2+ F－ Cl － Br － － SO I2 － SO4 H 3 2－ 2 PO － Ac 4 O－ N －

0

(b)

(c)

Fig. 4. (a) Fluorescence responses of DNBY (10 μM) to various biologically relevant species in PBS aqueous solution (C2H5OH/PBS = 3:7, pH 7.4). Black bars represent

ACCEPTED MANUSCRIPT the fluorescence response of the probe to some representative biological species (1 mM for K+, Na+, Mg2+, Ca2+, F−, Cl−, Br−, I−, SO42−, SO32−, H2PO4−, AcO−, N3−, 1O2, •OH, H2O2, GSH, Hcy, Cys). Red bars represent the subsequent addition of NaHS

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(500 μM) to the mixture. Spectra were recorded after incubation with different

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biologically relevant species for 15 min at room temperature. λex = 560 nm, λem = 635

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nm. Slits: 5/5 nm. (b) Color changes of DNBY (10 µM) in the presence of H2S (50.0 equiv.) and other biologically relevant species (100 equiv.) in PBS aqueous solution (C2H5OH/PBS = 3:7, pH 7.4). (c) Fluorescence photographs of DNBY (10 µM) in the

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presence of H2S (50.0 equiv.) and other biologically relevant species (100 equiv.) in

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8000

DNBY DNBY+NaSH DNBY+Cys DNBY+Hcy DNBY+GSH

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4000

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6000

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Fluorescence Intensity

PBS aqueous solution (C2H5OH/PBS = 3:7, pH 7.4).

2000

0

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0

200

400

600

800

Response Time (s)

Fig. 5. Fluorescence changes of DNBY (10 μM, C2H5OH/PBS = 3:7, pH 7.4) in the presence of Cys, Hcy, GSH, and NaHS, respectively. λex = 560 nm, λem = 635nm. Points represent the fluorescence intensity at 635 nm. Black points: DNBY, pink points: DNBY + Hcy (500 μM), green points: DNBY + GSH (500 μM), blue points: DNBY + Cys (500 μM), red points: DNBY + NaHS (500 μM). 3.5. Study of the reaction mechanism To demonstrate the sensing mechanism depicted in Scheme 2, the probe DNBY was treated with 50 equiv. of NaHS for 0.5 h, then the product was isolated by silica gel column for 1H NMR and HR-MS analysis. As shown in Fig. 6, the 1H NMR

ACCEPTED MANUSCRIPT spectrum of the probe after treatment with NaHS was identical to that of compound 3, which indicated that the dinitrophenyl group was removed by HS−. HR-MS spectra

H2S Reaction Site

OH

PET H2S N N B F F

N N B F F

O2N

N

N

O non-fluorescent

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N

+

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H+

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NO2 O SO O

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O2N

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also showed the same mass peak with compound 3 (shown in Fig. S2).

SH

+ SO2

NO2

NH O

strongly red fluorescent

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Scheme 2 The proposed sensing mechanism for H2S.

Fig. 6. (a) 1H NMR spectrum of the compound 3 in DMSO-d6. (b) 1H NMR spectrum of the isolated product from probe DNBY after treatment with NaHS in DMSO-d6.

ACCEPTED MANUSCRIPT 3.6. Cell imaging The ability of probe DNBY to sense H2S in living cells was examined by using confocal fluorescence microscopy. HeLa cells were co-incubated with DNBY (10 μM)

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for 30 min at 37 °C, and then washed with PBS to remove excess probe. As shown in Fig. 7a, HeLa cells showed no fluorescence in the absence of NaHS. When the HeLa

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cells were treated with 50 μM NaHS for 15 min, a clear cell profile with red fluorescence was observed, as shown in Fig. 7b. The fluorescence images in the red channel became brighter as the concentration of NaHS increased from 0 to 150 μM,

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verifying the fluorescence was induced by NaHS. The result indicates that the probe DNBY is membrane permeable and can report H2S in the living cells in a dose

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dependent manner. Moreover, the images in Fig. 7d also suggested that the probe has

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low toxicity since the cellular morphology was maintained.

(b)

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(a)

(c)

(d)

Fig. 7. Confocal fluorescence images of HeLa cells. Cells were incubated with probe DNBY (10 μM) for 30 min, and subsequently treated with (a) 0 μM NaHS, (b) 50 μM NaHS, (c) 150 μM NaHS for 15 min; (d) bright-field images. The images were acquired using confocal fluorescence microscope at 100× magnification. Scale bar: 20 μm.

ACCEPTED MANUSCRIPT 4. Conclusion In summary, we have successfully developed a novel fluorescent probe for H2S

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base on indole-BODIPY. The probe displayed a remarkable fluorescence “turn on”

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response (200-folds enhancement) to H2S, which was triggered by cleavage of the

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2,4-dinitrobenzenesulfonyl group. This probe has some advantages including fast response (t1/2 = 3 min), long emission wavelength (λem = 635 nm), good selectivity and low detection limit (26 nM). Cell staining results indicated that the probe could

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detect and visualize H2S in living cells.

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Acknowledgements

This work was financially supported by NSFC (No. 21203138, 31371750) and the

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Natural Science Foundation of Hubei Province (2013CFC007).

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Graphical abstract

ACCEPTED MANUSCRIPT Highlight ●

A far-red emitting probe for H2S has been developed based on indole-BODIPY

The probe responds to H2S (t1/2 = 2.5 min) rapidly with high sensitivity and

selectivity.

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The detection limit is as low as 0.02 μM for H2S.

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●

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platform.