Efficient Two-Photon Fluorescent Probe with Red Emission for

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Analytical Chemistry

An Efficient Two-Photon Fluorescent Probe with Red Emission for Imaging of Thiophenols in Living Cells and Tissues Hong-Wen Liu, Xiao-Bing Zhang*, Jing Zhang, Qian-Qian Wang, Xiao-Xiao Hu, Peng Wang, and Weihong Tan

Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082.

* To whom correspondence should be addressed.

E-mail: [email protected].

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ABSTRACT: Thiophenols, a class of highly toxic and pollutant compounds, are widely used in industrial production. While some aliphatic thiols play important roles in living organisms. Therefore, the development of efficient methods to discriminate thiophenols from aliphatic thiols is of great importance. Although several one-photon fluorescent probes have been reported for thiophenols, two-photon fluorescent probes are more favorable for biological imaging due to its low background fluorescence, deep penetration depth and so on. In this work, a two-photon fluorescent probe for thiophenols, termed NpRb1, has been developed for the first time by employing 2, 4-dinitrobenzene-sulfonate (DNBS) as a recognition unit (also a fluorescence quencher), and a naphthalene-BODIPY-based through-bond energy transfer (TBET) cassette as a fluorescent reporter. The TBET system consists of a D-π-A structured two-photon naphthalene fluorophore and a red-emitting BODIPY. It displayed highly energy transfer efficiency (93.5%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λem=586 nm), which is highly desirable for bioimaging applications. The probe exhibited a 163-fold thiophenol-triggered two-photon excited fluorescence enhancement at 586 nm. It showed a high selectivity and excellent sensitivity to thiophenols, with a detection limit of 4.9 nM. Moreover, it was successfully applied for practical detection of thiophenol in water samples with a good recovery, and two-photon imaging of thiophenol in living cells, and tissues with tissue-imaging depths of 90-220 µm, demonstrating its practical application in environmental samples and biological systems.

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INTRODUCTION Thiols including aliphaticthiols and thiophenols are an important class of molecules in biological system and chemical industry. The former including cysteine (Cys), homocysteine (Hcy), glutathione (GSH), et al, which play important roles in a wide range of biological functions.1-3 Whereas, thiophenols are a class of highly toxic and pollutant compounds,4 which are widely used in production of pesticides, pharmaceuticals, polymers, and dyes.5 The exposure to thiophenols liquid or vapor may induce sever central nervous system damage and other related system injuries, even death.6 The median lethal dose (LC50) for thiophenols ranges from 0.01 to 0.4 mM for fish,7 and thiolphenols have been added to the priority lists of pollutants by the United States Environment Protection Agency (EPA waste code: P014).8 Therefore, The establishment of a highly sensitive and selective method to discriminate thiophenols from aliphaticthiols is of great importance in the fields of chemical, environmental and biological sciences.

Towards sensing thiophenols, fluorescence method seems to be the best choice by virtue of its high sensitivity, non-destructive fast analysis and providing in situ and real-time information.9 In the past decade, great significant efforts have been paid toward the development of fluorescent probes for aliphaticthiols.10-12 Most of previous fluorescent probes can just discriminate aliphaticthiols such as cysteine, glutathione, and homocysteine from other amino acids.13-20 A probe being capable of distinguishing thiophenols over alphaticthiols was first developed by Wang et al in 2007.21 Since then, several fluorescent probes that can discriminate thiophenols from alphaticthiols have been reported.22-31 However, all of them are 3



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excited by one-photon light, their biological applications were limited because of the requirement of a rather short excitation wavelength (usually 120 GM) and many other attractive features, such as high fluorescence quantum yield and good photostability.35-39 However the emission wavelength of naphthalene derivatives is around 480 nm,32,33 which might limit its tissue-imaging depth.40 Through-bond energy transfer (TBET) might provide a good strategy to solve this problem, by employing a two-photon fluorophore as the donor and a fluorophore with emission wavelength in the red to NIR range as the acceptor. Unlike FRET system, in TBET system, the donor and the acceptor are directly connected by electronically conjugated bond, so they do not require a strong spectral overlap between the donor emission and the acceptor absorption. As a result, TBET system bears several advantages over FRET systems, including larger pseudo-stokes shifts and emission shifts, higher energy transfer efficiency.41-45 Our group previously proposed a naphthalene-rhodamine B TBET cassette, 4


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termed Np-Rh, which exhibited two well-resolved emission peaks (separated by 100 nm) and a highly energy transfer efficiency. It also showed a target-modulated ratiometric two-photon fluorescence response, and was successfully applied for two-photon imaging of Cu2+ and Pd2+ in living cells and tissues.46, 47

Herein, we reported a novel naphthalene-BODIPY TBET cassette (NpRb, Scheme 1) for design of a two-photon fluorescent probe for highly sensitive and selective detection of thiophenols based on photoinduced electron transfer (PET) mechanism. We used a classic two-photon naphthalene dye as a donor and a red emission BODIPY derivative as an acceptor. A minimal spectral overlap was observed between the emission of naphthalene derivative and the absorption of BODIPY derivative. The cassette displayed highly energy transfer efficiency upon one-photon excitation at 420 nm or two-photon excitation at 780 nm. For the probe molecule, the fluorescence of the TBET cassette was quenched by the recognition unit DNBS. The introduction of thiol compounds could induce the cleavage of the probe molecule through a SNAr process, and released the fluorophore to result in a turn-on fluorescence signal. The probe showed both one-photon and two-photon excited fluorescence enhancement response to thiophenols in aqueous solutions with a detection limit of 4.9 nM and a high selectivity. Most importantly, it was successfully applied for detection of thiophenol in water samples and for two-photon imaging of thiophenol in living cells and tissues, with satisfactory results.

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Br O

N

N

N N

Donor 1 N

N O

O

ArSH Br

N F

B

N B

N

N

N F F

F

F

O O S O

Acceptor 2

NpRb1

OH

B

N F

NO2

O2 N

NpRb

OH

Scheme 1. Structures of donor, acceptor, probe NpRb1; and the response mechanism of probe NpRb1 to thiophenols. EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were purchased from commercial suppliers and used without further purification. Water was purified and doubly distilled by a Milli-Q system (Millipore, USA). The one-photon excited fluorescence measurements were conducted at room temperature on a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ) with both excitation and emission slit set at 3.0 nm. Mass spectra were performed using an LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). NMR spectra were recorded on a Bruker DRX-400 spectrometer using TMS as an internal standard. All chemical shifts are reported in the standard δ notation of parts per million. Thin layer chromatography (TLC) was conducted using silica gel 60 F254, and column chromatography was carried out over silica gel (200-300 mesh), both of them were obtained from Qingdao Ocean Chemicals (Qingdao, 6


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China). The pH was measured with a Mettler-Toledo Delta 320 pH meter. Two-photon fluorescence images of Hela cells and tissues were obtained using an Olympus FV1000-MPE multiphoton laser Scanning confocal microscope (Japan). Compound NpRb1 was efficiently synthesized following the synthetic methodology shown in Scheme 2.

N Br Br

O O O B B O O

N

O

B

O

N N

N

O

1

N

B

F

F

N

HO

N

B

F

F

CHO

3

OH

N

B

N

N

CHO

N

F

F

4 N

Br

O Cl S O

N O

O

NO2 NO2

N

B

F

N N

F

N NB F

2

F

B

N F

F OH

NpRb1

O OS O O2N

NO2

NpRb

OH

Scheme 2. Synthetic route for probe NpRb1.

Synthesis of compound 3. 4-Br-BODIPY was synthesized according to a literature procedure.48 400 mg of 4-Br-BODIPY (1mmol), 358 mg (1.5 eq.) of bis(pinacolato), 150 mg (1.5 eq.) of potassium acetate and 20 mg (0.025 eq.) of Pd(dppf)Cl2 were refluxed in 100 mL toluene under N2-atmosphere for 12 h. After cooling to room temperature, the solution was

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filtered, and the solid was washed with CH2Cl2. The combined organic solution was then concentrated under reduced pressure. Further purified by the silica gel chromatography (ether /ethyl acetate, 20:1, v/v) gave compound 3 as a orange-yellow solid (306 mg, 68% ). 1HNMR (CDCl3, 400 MHz) δ (ppm): 7.93(d, J = 7.92Hz, 2H), 7.32 (d, J = 7.31Hz, 2H), 5.98 (s, 2H), 2.56 (s, 6H), 1.40 (s, 12H), 1.38 (s, 6H). MS (EI): m/z 451.2, (M+), calcd 452.34.

Synthesis of compound 4. Compound 1 was synthesized according to our previous work.46,47 Compound 3 (450 mg, 1 mmol), compound 1 (330 mg, 0.9 eq.), potassium acetate (150 mg, 1.5 eq.), and Pd(dppf)Cl2 (20 mg, 0.025 eq.) were refluxed in 100 mL toluene/H2O (50:1, v/v) mixed solution under N2-atmosphere for 12h. The solvent was concentrated under reduced pressure. After purified by the silica gel chromatography (petroleum ether /ethyl acetate, 10:1, v/v), compound 4 was obtained as a orange-yellow solid (326 mg, 53%). 1

HNMR (CDCl3, 400 MHz) δ (ppm): 8.67(s, 1H), 8.25 (d, J = 8.24, 1H), 7.91-7.81(m, J =

7.86, 8H), 7.70(dd, J = 7.69, 1H), 7.41 (d, J = 7.40, 2H), 6.01 (s, 2H), 3.15 (s, 6H), 2.58 (s, 6H), 1.45 (s, 6H). MS (ESI): m/z 611.4 [M+H]+, 633.1 [M+Na]+ , calcd 610.5.

Synthesis

of

compound

NpRb.

Compound

4

(305

mg,

0.5

mmol),

4-Hydroxy-benzaldehyde (61 mg, 0.5 mmol) and piperidine (1 mL) was dissolved in dry toluene (100 mL) in a Dean-Stark apparatus and then refluxed for 12 h. The solvent was concentrated under reduced pressure. The residue was purified by the silica gel chromatography (petroleum ether /ethyl acetate, 5:1, v/v), which gave compound NpRb as a dark-purple solid (46mg, 13%). 1HNMR (DMSO-d6, 400 MHz) δ (ppm): 10.00(s, 1H), 8.64(s, 1H), 8.24(s, 1H), 8.12 (dd, J = 8.11, 1H), 8.05 (dd, J = 8.02, 3H), 7.88(m, J = 7.86, 3H), 8


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7.55(d, J = 7.54, 2H), 7.50 (m, J = 7.48, 3H), 7.35 (m, J = 7.32, 2H), 6.21 (s, 1H), 3.09 (s, 6H), 2.54 (s, 3H), 1.52 (s, 3H), 1.47 (s, 3H). MS (ESI): m/z 713.4 [M-H]+ , calcd 714.6.

Synthesis of compound 2. Compound 2 was synthesized following the similar procedure of compound NpRb. 1HNMR (DMSO-d6, 400 MHz) δ (ppm): 9.98(s, 1H), 7.79(d, J = 7.78, 2H), 7.53(d, J = 7.51, 1H), 7.47(d, J = 7.46, 2H), 7.41(d, J = 7.40, 2H), 7.33(d, J = 7.31, 2H), 6.94(s, 1H), 6.87 (d, J = 6.86, 2H), 6.19 (s, 1H), 2.49 (s, 3H), 1.44 (s, 3H), 1.40 (s, 3H). MS (ESI): m/z 507.1 [M-H]+ , calcd 508.1.

Synthesis of compound NpRb1. Compound NpRb (35mg, 0.05mmol) was dissolved in CH2Cl2 and added Et3N (6mL) followed with 2, 4-Dinitrobenzenesulfonyl Chlorid (26mg, 0.1mmol). After being stirred at room temperature for 2 h, the solvent was removed under reduced pressure, and the residue was purified by the silica gel chromatography ( petroleum ether /ethyl acetate, 8:1, v/v ) to afford compound NpRb1 as a dark-red solid (31mg, 66%). 1

HNMR (DMSO-d6, 400 MHz) δ (ppm): 8.62 (s, 1H), 8.27 (d, J = 8.26, 2H), 8.22 (s, 1H),

8.12 (d, J = 8.11, 1H), 8.05 (d, J = 8.04, 2H), 8.01 (d, J = 8.00, 1H), 7.87 (m, J = 7.85, 3H), 7.76 (d, J = 7.75, 1H), 7.66-7.53 (m, J = 7.59, 5H), 7.49 (s, 1H), 7.37 (dd, J = 7.34, 2H), 7.27 (d, J = 7.26, 2H), 7.02 (s, 1H), 6.98 (d, J = 6.96,1H), 6.28 (s, 1H), 3.09 (s, 6H), 2.51 (s, 3H), 1.51 (s, 3H), 1.48 (s, 3H). HRMS (ESI): m/z 945.2797 [M+H]+, m/z 967.2656 [M+Na]+ , calcd 944.2611.

Spectrophotometric Experiments. Both the fluorescence and UV-Vis absorption measurement experiments were carried out in 10 mM phosphate buffered solution containing

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50% EtOH as the co-solvent. The fluorescence emission spectra were recorded at an excitation wavelength of 420 nm with emission wavelength ranged from 440 to 700 nm. A 1×10-3 M stock solution of probe NpRb1 was prepared by dissolving compound NpRb1 in EtOH. Stock solutions of C6H5SH, p-CH3-C6H4SH, o-NH2-C6H4SH, C6H5OH, C6H5NH2 were prepared in EtOH (10 mM, respectively). The solutions of various testing species were prepared from GSH, Cys, Hcy, glucose, H2O2, NaClO, NaHS, NaN3, NaI, MgCl2, CaCl2 and Zn(NO3)2 in twice-distilled water. The test solution of the NpRb1 (5µM) in 2 mL of 10 mM PBS buffer EtOH solution (pH 7.4) was prepared by placing 10 µL of the NpRb1 stock solution (1×10-3 M) in 1.99 mL of the various analytes buffer/EtOH solution (by adding EtOH to the resulting solution to keep the ratio of organic phase at 50%). The resulting solutions were kept at ambient temperature for 30 min and then the fluorescence intensities were measured. The two photon excited fluorescence intensity was measured at 700-900 nm by using Rhodamine B as the reference, whose two-photon property has been well characterized in the literature.49 NpRb1 and NpRb were dissolved in PBS buffered EtOH solution (10 mM, pH 7.4, containing 50% EtOH), then the intensities of the two-photon-induced fluorescence spectra of the samples and reference excited at the same wavelength were determined. The two-photon absorption cross-section (δ) was calculated by using the following formula ( 1 ):

δ=δr(SsΦrфrCr)/(SrΦsфsCs)

(1)

where the subscripts s and r denote the sample and reference molecule, respectively.39

Detection of Thiophenol in Water Samples. Water samples were collected from Xiang River (Changsha, China). The water samples were filtered with pH adjusted to 7.4 prior to 10


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use. The thiophenol stock solution at different concentration was spiked in these samples, and the probe NpRb1 was then added to detect its concentration, with analytical results shown in Table 1.

Cytotoxicity Study and Two-photon fluorescence microscopy imaging of thiolphenol in Hela cells. To study the cytotoxicity, Hela cells were seeded at 1×105 cells per well in 96-well plates and incubated for 24 h before treatment, followed by exposure to different concentrations (2-16 µM) of probe NpRb1 and NpRb for an additional 24 h or 48 h. And then the cytotoxic effects of NpRb1 and NpRb were determined using MTT assays. The absorbance value at 570 nm was measured by a microplate reader. To investigate the capability of probe NpRb1 for detection thiolphenol in living cancer cells, Hela cells were washed with Dulbecco's phosphate buffered saline (DPBS), followed by incubating with 5 µM of NpRb1 for 30 min (in DPBS containing 1% DMSO), then by washing with DPBS three times. After the cells were incubated with 5 µM of thiophenol (in DPBS containing 1% DMSO) for another 30 min at 37 °C, the Hela cells were washed with DPBS three times and imaged. For control experiments, the cells were pretreated with 5 µM NpRb1 only or pretreated with N-methylmaleimide (NMM, 100 µM, a thiols scavenger) and then thiophenol (5 µM). Confocal fluorescence imaging of Hela cells was then performed using an Olympus FV1000 laser confocal microscopy (Japan). Two-photon fluorescence microscopy images of Hela cells were carried out by exciting the probes with a mode-locked titanium-sapphire laser source set at wavelength 780 nm, the emission wavelengths were recorded at 470-530 and 550-650 nm respectively. 11



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Two-Photon Fluorescence microscopy imaging of thiolphenol in Liver Tissue Slices from Nude Mice. Frozen tissue slices were prepared from the livers of nude mice. The slices were incubated with 10 µM NpRb1 at 37 °C for 1h then washed with DPBS three times, and cultured with 50 µM thiophenol for another 1h at 37 °C. Finally, the slices were washed with DPBS three times, and then two-photon fluorescence microscopy images were collected. The excitation wavelength of the femtosecond laser was set at 780 nm, the emission wavelengths were recorded at 470-530 and 550-650 nm, respectively.

RESULTS AND DISCUSSION Optimized Design and Synthesis of the fluorescent probe. Although several one-photon excited fluorescent probes have been constructed for thiophenols, to our best knowledge, no two-photon fluorescence probe for discrimination of thiophenols from aliphaticthiols has been developed so far. In this paper, we constructed a two-photon fluorescence probe with red emission for thiophenols, since fluorescent probes with emission wavelength in the red to NIR range exhibit deeper imaging penetration depths. In our design, TBET mechanism was chosen to obtain red fluorescence emission with a highly efficient energy transfer, as it does not need spectral overlap between the donor and acceptor (see Figure S1 in Supporting Information). We chose naphthalene derivative as the TBET system energy donor for its outstanding two-photon properties, and a well reported red-emitting styryl-BODIPY was chosen as the energy acceptor.50,51 Such a design might endow the TBET system a large pseudo-stokes shift and emission shift, both one-photon and two-photon excited red-emission. A strongly electron-withdrawing BNDS group, which is widely used for the protection of an amino or 12


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hydroxy group, was incorporated into the TBET cassette as both a recognition unit for thiophenols and a fluorescence quencher, as the incorporation of DNBS moiety into the TBET cassette would quench its fluorescence through a PET process. In the presence of thiophenols, the fluorescence of the probe should be recovered due to the effective thiolysis of DNBS group. The synthetic methodology for NpRb1 is outlined in Scheme 2 and its structure was confirmed by 1HNMR and MS.

140 120 100

F/F0

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80 60 40 20 0 0

5

10

15

20

Time (min)

Figure 1. Time-dependent fluorescence spectral changes of NpRb1 with thiophenol (probe 5 µM, thiophenol 5 µM) in PBS buffered (10mM, pH=7.4) aqueous EtOH solution (1:1, v/v). Time points represent 0, 0.17, 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 5, 6, 8, 10, 15, and 20 min, λex=420. The inset show the visual fluorescence color of NpRb1 (10 µM) before (left) and after (right) incubation with thiophenol for 30 min (UV lamp, 365 nm). Fluorescent Analytical Performance of NpRb1. The fluorescence spectra of NpRb1 (5 µM) in the absence and presence of thiophenol were first recorded in buffered (10 mM PBS, pH = 7.4) aqueous EtOH solution (H2O/EtOH = 1:1, v/v). After a few minutes, a dramatic fluorescence enhancement at 586 nm was observed upon the addition of thiophenol (Figure 1), and the fluorescence intensity got saturation after 20 min. The changes of the fluorescence spectra of NpRb1 (5 µM) upon the gradual addition of thiophenol are further investigated 13



(Figure 2a). Upon the addition of thiophenol, a characteristic fluorescence emission peak at 586 nm was observed, which belonged to the acceptor, and its intensity increased linearly with the concentration of thiophenol ranging from 50 nM to 2 µM (Figure 2b). The detection limit for thiophenol was calculated to be 4.9 nM (3σ/slope), which is lower than other previously reported one-photon fluorescence probes.21,23-28 The fluorescence intensity of the donor remained very weak, which indicted a high energy transfer efficiency (ETE) between the donor and the acceptor. The ETE was determined to be 93.5% (see Figure S1 in Supporting Information). This data was consistent with previous results (due to TBET system is always accompanied by FRET, the ETE is limited to be less than 100%).45-47 (a) 200

150

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400000

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300000 0 0

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100000

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(b) 100

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40

10

20 0 0.0

0 0.0

0.5

1.0

ArSH(µM)

1.5

0.5

2.0

ArSH(µM)

Figure 2. (a) The fluorescence emission spectra of NpRb1 (5µM) in the presence of different concentrations of thiophenol (0, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 1.2, 1.5, 2, 3, 5, 10, 12, 16, 20 µM) in PBS buffered (10mM, pH=7.4) aqueous EtOH solution (1:1, v/v). The inset shows calibration curve of NpRb1 to thiophenol, the curve was plotted with the fluorescence intensity at 586 nm vs thiophenol concentration after incubation of them for 30 min. (b) The linear responses at low thiophenol concentrations. λex=420 nm. Selectivity and Effect of pH. Selectivity is another quite important parameter to evaluate the performance of a new fluorescent probe. Probe NpRb1 was then treated with a variety of potentially competing species to evaluate the selectivity, including thiols, other nucleophile, reactive oxygen species, metal ions and some biological molecules, with results given in Figure 3 (black bar). Considering the actual concentration of GSH is 1~10 mM in living cells, a concentration of 15 mM was chosen for GSH to investigate its effect on the response of the probe to thiophenol. As shown in figure 3, in the presence of thiophenol, 4-methylthiophenol and 2-aminobenzenethiol, the fluorescence intensity was significantly enhanced at 586 nm, but the intensity of 2-aminobenzenethiol was smaller than thiophenol or 4-methylthiophenol. 15



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The possible reason is that the ortho amino-group of the thiol might result in steric hindrance, thus the reactivity of the SNAr reaction is decreased. As expected, only Cys, GSH and Hcy caused a slight fluorescence intensity enhancement, while other non-thiophenols species did not induce any observable spectral changes. To further demonstrate the selectivity of probe NpRb1, the specificity of the probe towards thiophenol in the presence of other competing species was also investigated. The results showed in the figure 3 (red bar), probe NpRb1 remained essentially unaffected in the presence of other species. Accordingly, it was easy to conclude that there was no interferences from other competing species towards the probe. These data demonstrated that probe NpRb1 was suitable for detection of thiophenols in practical and biological samples.

The effect of pH on the fluorescence intensity of NpRb1 in the absence and presence of thiophenol were also investigated (Figure S3 in Supporting Information). Experimental results indicated that NpRb1 was pH insensitive in pH range from 2.5 to 11.5. As expected, in the presence of thiophenol, the fluorescence intensity increase with the pH ranging from pH 2.5 to 9, due to the enhanced ionization of thiophenol to result in a faster SNAr process. With pH>10, the fluorescence intensity decreased with the pH increase from pH 10.0 to 11.5. We thought this was induced by the PET process from phenolic moiety to the fluorophore. The results demonstrated that the probe could work effectively at pH 7.4, and the probe was favorable for applications in biological samples.

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40

0 0 1 2 3 4 5 6 7 8 9 101112131415161718

Figure 3. Fluorescence responses of NpRb1 (5 µM) toward thiols (3 µM) and other substances (15 mM for GSH and 0.1 mM for others) in PBS buffered (10 mM, pH=7.4) aqueous EtOH solution (1:1, v/v) at room temperature for 30min. The fluorescence measurement at λex=420nm. The fluorescence intensity at λem=586 nm was plotted versus substances: (1) blank, (2) thiophenol, (3) 4-methylthiophenol, (4) 2-aminobenzenethiol, (5) Cys, (6) GSH, (7) Hcy, (8) H2S, (9) NaClO, (10) H2O2, (11) phenol, (12) aniline, (13) NaN3, (14) NaI, (15) MgCl2, (16)CaCl2, (17) Zn(NO3)2, (18) glucose. The black bar represents the fluorescence intensity of only a single analyte with probe. Red bar represents the fluorescence intensity of only a single analyte and thiophenol with probe. TP Fluorescence Properties of NpRb1. The maximum Φδ value of NpRb is estimated to be 116.45 GM (1 GM = 10 50 (cm 4 s)/photon) at 780 nm (Figure 4a). In addition, excited of −

NpRb at 780 nm resulted in emission mainly from the acceptor with a peak at 586 nm and a minor emission peak at 480 nm (Figure 4b). The results demonstrated that the two-photon excited TBET process was truly occurred with a high ETE. A 163 fold TP excited fluorescence enhancement at 586 nm was observed between probe molecule NpRb1 and thiophenol-triggered cleaved product NpRb when excited by 780 nm femtosecond pulses (Figure 4b). 17



(a) (b)

120

40000

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35000

dF (GM)

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80 60 40 20 0 700

750

800

850

30000 25000 20000 15000 10000 5000 0 400

900

450

500

550

600

650

Emission (nm)

Wavelength(nm)

Figure 4. (a) TP absorption cross-section of NpRb (●) and NpRb1(○). (b) TP excited emission spectra of donor 1 (black line), acceptor 2 (red line), NpRb (blue line) and NpRb1 (green line). TP excitation at 780 nm in PBS/EtOH (1:1, v/v, 10mM, pH=7.4). Response Mechanism. Similar to previously reported DNBS-based thiolphenol probes, the fluorescence enhancement response of NpRb1 was via a thiolate anion induced SNAr process which resulted in the cleavage of the DNBS group from the probe molecule (Scheme 1). To verify the proposed mechanism, the change of the UV-vis spectra for NpRb1 upon the addition of thiolphenol was investigated (Figure 5a). The free NpRb1 exhibited two maxima absorption peak at 396 nm and 564 nm respectively. The introduction of thiolphenol could induce obvious intensity enhancement at 570 nm and it showed a slightly red-shift, due to deprotection of hydroxyl groups and increased the intramolecular charge transfer (ICT) feature of NpRb. We also measured the UV-vis spectra of NpRb, it showed similar UV-vis spectra as that of the reaction product of NpRb1 with thiolphenol. To further verify our hypothesis, the purified product of the reaction of NpRb1 with thiolphenol was then characterized by HPLC (Figure 5b), which agreed well with the pre-synthesized compound

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NpRb, directly indicating the correct of our proposed mechanism (HPLC analysis procedure see in Supporting Information).

(a)

Figure 5. (a) UV−vis spectra of NpRb1 (5 µM), NpRb (5 µM) and the reaction product of NpRb1 (5 µM) with thiophenol (20 µM) after incubation of them for 1 h in PBS buffered (10 mM, pH=7.4) aqueous EtOH solution (1:1, v/v); (b) HPLC of NpRb (10 µM), NpRb1 (10 µM), and reaction product of NpRb1 (10 µM) with thiophenol (50 µM) after incubation of them for 1 h in PBS buffered (10 mM, pH=7.4) aqueous EtOH solution (1:1, v/v). The bleak line, red line and blue line represent NpRb1, NpRb and reaction product of NpRb1 with thiophenol respectively. Detection of Thiophenols in Water Samples. To evaluate the practicality of this new probe, we employed probe NpRb1 to detect the thiophenol concentrations in water samples from Xiang River. The water samples were spiked with different thiophenol concentrations (0.1 µM, 0.5 µM, and 1 µM), with the results shown in the Table 1. It was observed that the results obtained for river water samples shown good recovery values, which demonstrated that the proposed probe was capable of practical thiophenols detection.

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Table 1. Determination of Thiophenol Concentrations in Water Samples. thiophenol spiked (M)

thiophenol determined (M) meana ± SDb

Recovery (%)

River water 1

0.0

Not detected

--

River water 2

1.0×10-7

（9.58±0.07）×10-8

95.8

River water 3

5.0×10-7

（5.12±0.05）×10-7

102.4

River water 4

1.0×10-6

（9.75±0.04）×10-5

97.5

Sample

a

Mean of three determinations.

b

SD: standard deviation.

TP Bioimaging of thiolphenol in Living Cells and Liver Tissues. Both the high selectivity and sensitivity of probe NpRb1 towards thiolphenol and a dramatic TP excited fluorescence enhancement after addition of thiolphenol demonstrated that the probe was especially favorable for TP bioimaging thiolphenol in biological samples. The cytotoxicity of the probe NpRb1 and NpRb was first evaluated by MTT assays for HeLa cells. We incubation the Hela cells with different concentrations (2-16 µM) of probe NpRb1 and NpRb for 24 h or 48 h respectively, and experimental results showed that both the NpRb1 and NpRb were almost no cytotoxicity to HeLa cells even with a high concentration of 16 µM (Figure S4). So, we further employed the probe NpRb1 to detect thiolphenol in living cells, the results were shown in figure 6. The HeLa cells were incubated with NpRb1 only exhibited no obvious fluorescence signal in both the green channel and the red channel (Figure 6c, d), due to the strong PET process of the probe. But after incubation of cells with 5 µM thiolphenol, a significant fluorescence enhancement was observed in the red channel, and as expected, very 20


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weak fluorescence signal in the green channel (Figure 6e, f), corresponding to strong fluorescence from the acceptor and the weak fluorescence from the donor. The cells treated with donor 1 only showed a strong fluorescence signal (Figure 6a), and when cultivated with acceptor 2, a weak fluorescence signal was observed (Figure 6b). These results demonstrated the effective TBET process of the probe and dramatically fluorescence enhancement induced by thiolphenol. In another control experiment, the Hela cells were first incubated with N-ethylmaleimide (0.1 mM, NEM, a scavenger of thiols), and then treated with probe NpRb1 (5 µM), and finally pretreated with thiolphenol (5 µM). The results (Figure 6g, h) suggested that the intracellular fluorescence enhancement was indeed triggered by thiophenol. All these results demonstrated that NpRb1 is a membrane permeable two-photon fluorescence probe and suitable for imaging of thiolphenol in biological samples.

Figure 6. TP confocal microscopy images of 5µM donor 1, acceptor 2 and NpRb1 in live HeLa cells. TP images of donor 1 ( a ) and acceptor 2 ( b ). TP images of NpRb1 from the green channel ( c ) and the red channel ( d ) without thiopheonol. And TP images of NpRb1 from the green channel ( e ) and the red channel ( f ) with thiopheonol. TP images from the 21



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green channel ( g ) and the red channel ( h ) with both NEM and thiopheonol. λex=780nm, Green channel: λem= 470-530 nm; red channel 550-650 nm. Scale bar: 30 µm. To further show the outstanding advantage of our red emission two-photon fluorescence probe in TP imaging, TP excited imaging of NpRb1 for thiolphenol in rat liver tissue slices was then carried out (Figure 7), with donor 1 (D-π-A structured two-photon naphthalene dye) and acceptor 2 as controls. The changes of fluorescence signal intensity with scan depth were recorded by TPFM in the Z-scan mode (Figure S5, Supporting Information). The results showed that the probe was clearly capable for deep tissue TP imaging, with an imaging depths of 90-220 µm observed, and the donor 1 could applied for tissue TP imaging at a depth of 57.5-152.5 µm, while acceptor 2 exhibited almost no TP fluorescence signal. It was easy to conclude that the probe had excellent tissue penetrating and staining abilities, and the tissue imaging depth of the probe was obviously larger than the typical D-π-A structured two-photon naphthalene dye. a

137.5µm

b

137.5µm

Figure 7. TP imaging of a rat liver frozen slice stained with 10 µM NpRb1 at 137.5 µm for 1h, and then treated with 50 µM thiopheonol for another 1 h. λex=780 nm, Green channel: λem= 470-530 nm; red channel 550-650 nm. Scale bar: 100 µm.

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CONCLUSIONS In summary, we have reported a red emission two-photon fluorescent probe based on TBET strategy for detection of thiophenols. A two-photon D-π-A-structured naphthalene derivative and a red emitting BODIPY derivative are directly connected by conjugated bond to form a TBET probe, the probe showed high energy transfer efficiency, large pseudo-Stokes shifts upon one-photon excitation and red fluorescence emission, which make the probe more favorable for bioimaging applications. Besides, the probe NpRb1 exhibited a robust and special response to thiophenol with a large signal-to-background ratio both in vitro and in vivo, due to the thiophenols-mediated cleavage of the strong fluorescence quencher DNBS through the SNAr process. The probe has been successfully applied for detection of thiophenol in water samples. Furthermore, we demonstrated that this novel probe was very desirable for TP imaging of thiophenols in living cells and tissue with improved imaging results compare with previously reported one-photon thiophenols probe because of exciting with NIR laser pulses and a red fluorescence emission. All these features demonstrate that probe NpRb1 is promising for practical application in biological systems and environmental systems.

ACKNOWLEDGEMENT This work was supported by the National Key Scientific Program of China (2011CB911000), the National Key Basic Research Program of China (2013CB932702), NSFC (Grants 21325520, 21327009, J1210040, 21177036), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), National Instrumentation Program (2011YQ030124), and Hunan Provincial Natural Science Foundation (Grant 11JJ1002). 23



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SUPPORTING INFORMATION AVAILABLE Supplementary spectral data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Efficient Two-Photon Fluorescent Probe with Red Emission for

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