Highly Selective and Sensitive 1-Amino BODIPY-Based Red

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Highly selective and sensitive 1-amino BODIPY-based red fluorescent probe for thiophenols with high off-to-on contrast ratio Xiangmin Shao, Ruixue Kang, Yuanlin Zhang, Zhentao Huang, Fangfang Peng, Jian Zhang, Yue Wang, Fuchao Pan, Weijuan Zhang, and Weili Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5028947 • Publication Date (Web): 28 Nov 2014 Downloaded from http://pubs.acs.org on December 1, 2014

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Analytical Chemistry

Highly selective and sensitive 1-amino BODIPYbased red fluorescent probe for thiophenols with high off-to-on contrast ratio Xiangmin Shao,a Ruixue Kang,a Yuanlin Zhang,a Zhentao Huang,a Fangfang Peng,a Jian Zhang,b Yue Wang,a Fuchao Pan,a Weijuan Zhang*a and Weili Zhao*ab a

Key Laboratory for Special Functional Materials of the Ministry of Education, Henan

University, Kaifeng, 475004, P. R. China. E-mail: [email protected]. Tel/Fax: +86-37123881358. b

School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China. E-mail:

[email protected]. Tel/Fax: +86-21-51980111.

ACS Paragon Plus Environment

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ABSTRACT A highly selective and sensitive turn-on red fluorescent 1-amino BODIPY-based probe with high off-to-on contrast ratio has been developed. The probe displayed selective response to thiophenols over aliphatic thiols. Probe 1 is promising for the quantitative detection of thiophenol with linear response from 6 × 10−6 M to 1 × 10−4 M and the detection limit for PhSH reaches 4 × 10−6 M measured in acetonitrile/PBS buffer. The detection limit could be improved to 37 nM (detection limit to 4 ppb) in water when 1% Tween 20 was used to assist the dissolvation of probe 1 in water. Probe 1 is also a useful fluorescent probe for detecting thiophenols in living cells in red emission which may greatly improve the detectable sensitivity. KEYWORDS fluorescent probe, BODIPY, thiophenol, red fluorescent.


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INTRODUCTION Thiols are an important class of molecules in biological systems and chemical industry. Low molecular weight bioactive aliphatic thiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play important roles in living organisms, and are involved in a number of physiological processes.1-8 On the other hand, aromatic thiols (thiophenols) widely employed in the production of pesticides, polymers, and pharmaceuticals, are fairly toxic with a median lethal dose (LC50) of 0.01−0.4 mM for fish and are responsible for damaging Central Nervous System (CNS), kidney and liver functions on long-term explosure.9-12 The exposure to thiophenols results in symptoms like coughing, headache, nausea, burning sensation vomiting and even death.13-17 Thiophenols, in spite of their broad synthetic utility, have been listed as one of the prioritized pollutants by the United States Environmental Protection Agency.17 Therefore, a detection technique that can selectively differentiate toxic thiophenols from biologically important aliphatic thiols is of considerable significance in the fields of chemical, biological and environmental sciences. Due to close chemical properties of aliphatic thiols and thiophenols, most of probes utilizing the strong nucleophilicity of thiols exhibit poor selectivity.18-21 2,4dinitrobenzene sulfonyl (DNBS) was firstly used by Maeda et al. as sulfonate ester of a fluorophore for the detection of thiols.22 The high level of electron-deficiency enables DNBS moiety to act as an electron sink and incur photoinduced electron transfer (PET) resulting in the quenching of the fluorescence. The DNBS ester can easily undergo de-sulfonylation in the presence of thiols through nucleophilic aromatic substitution (SNAr) mechanism, releasing SO2 gas and the attached fluorophore, thus resulting in a fluorescence increase. Since the 2,4dinitrobenzenesulfonamide functionality was firstly reported for the detection of thiophenols by Wang’s group in intramolecular charge transfer (ICT) system,23 a number of fluorescent probes


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to discriminate thiophenols from aliphatic thiols with relatively high sensitivity and selectivity have been reported.24-35 Due to the difference of pKa values between thiophenols (ca. 6.5) and aliphatic thiols (ca. 8.5), the high degree of dissociation of thiophenols in a neutral reaction medium results in the predominant generation of the corresponding thiolate which can effectively react with 2,4-dinitrobenzenesulfonamide.23,24 However, these probes are mostly excited and/or emit in the UV to green region of the spectrum. We are interested in red fluorescent probe with high selectivity and sensitivity. Long-wavelength probes with emission in the red to near-infrared are optimal for imaging applications due to decreased light scattering, reduced autofluorescence, and increased optical transparency. Among the reported thiophenol probes, Ru (II) complexes and indole-based boron-dipyrromethene (BODIPY) emit in red region, however the off-to-on ratio is not optimal.26,29 A “brighter” and more sensitive sensor for thiophenols is highly desirable from the practical application standpoint of view. Among many classes of the fluorophores, BODIPY dyes have been recognized as the promising ones for the construction of molecular sensors because of their outstanding characteristics, such as high molar absorption coefficients, intense fluorescence quantum yield, valuable photo- and chemo-stability, and exceptional insensitivity to the polarity of solvents as well as to pH.36-42 Furthermore, various methods to access long-wavelength dyes have been developed, including extension of πconjugation, introduction of electron-donating substituent,43-47 conformation restriction, and incorporation of a nitrogen atom (Aza-BODIPY) in the skeleton.48,49 A few of NIR BODIPY/Aza-BODIPY dyes have been reported in our previous work.50-54 BODIPY was reported to be modified by introducing an efficient recognition site at the 2,3,5,6, or 8-position of the dipyrromethene core.55-57 However, only a few reports have been published through modification on the aromatic group at the 1-, or 7-position of BODIPY dye as signaling site.58-60


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Herein, we report a novel red fluorescent probe based on 1-amine substituted BODIPY for discrimination of thiophenols over aliphatic thiols sensitively.

Scheme 1. The design concept of fluorescent probe 1 for thiophenols and crystal structures of the related BODIPY species. In this paper, 1-NH2 BODIPY 2 as a fluorophore with high quantum yield and a DNBS group as recognition unit were combined to achieve a sensitive and selective fluorescent probe 1 which has no fluorescence due to photoinduced electron transfer (PET) pathway from the BODIPY to the DNBS moiety. The masked sulfonamide moiety can be facilely removed by a highly nucleophilic thiolate anion through SNAr process (Scheme 1).61 EXPERIMENTAL SECTION General Information. All reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. Acetonitrile in chromatographic purity and deionized water were used in detection. 1H NMR spectra were recorded on a VARIAN Mercury 400 MHz spectrometer. 1H NMR chemical shifts (δ) are given in ppm (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) downfield from Me4Si, determined by chloroform (δ = 7.26 ppm) and dimethyl sulfoxide (δ = 2.5 ppm). 13C NMR spectra were recorded on a VARIAN Mercury 100 MHz spectrometer. 13C NMR chemical


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shifts (δ) are reported in ppm with the internal CDCl3 and d6-DMSO at δ 77.0 and 39.4 ppm as standard, respectively. Mass spectrometric measurements were performed by the mass spectrometry service of Institute of Organic Chemistry, Chinese Academy of Sciences on a Bruker Reflex MALDI as matrix (20 kV). Fluorescence spectra were recorded on FluoroSENS spectrophotometer. UV/Vis spectra were recorded on Perkin-Elmer Lambda 35 UV/Vis spectrophotometer at room temperature. All spectra were recorded at room temperature except for the fluorescence microscopy images. Cell culture and fluorescence imaging. Mogic AE31 inverted biologic microscope was used for the images and the green channel was employed for the fluorescence imaging. Probe 1 (20 µM, 2 mL) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) was added to Human Hepatoma SMMC-7721 cells in a six-compartment cell culture plate that contained 2.0 mL culture medium, and was incubated at 37 °C for 30 min. After removing the culture medium and washing with PBS twice, the fluorescence images of cells were taken. PhSH (100 µM, 2.0 mL) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) was then added to the 7721 cells, which were further incubated at 37 °C for 30 min. After washing with PBS twice, the fluorescence images of cells were taken. Detection of PhSH with probe 1 in Water. Small amount of probe 1 (1.55 mg) was dissolved in

THF, then 1 mL Tween 20 was added to the mixture above. The THF in the resulting mixture were removed under reduced pressure to prepare the stock solutions. The solution was diluted and added to 100 mL volumetric flask with H2O. In all cases, the concentration of Tween 20 in H2O was maintained to be 1 %. The absorption and emission of probe 1 (10 µM) were followed upon addition of PhSH in acetonitrile (minimum amount).


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Synthesis.

7-methoxy-4,5-dihydro-1H-benzo[g]indole

(7),

7-methoxy-4,5-dihydro-1H-

benzo[g]indole-2- carbaldehyde (6) and 7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indole (4) were prepared according the reported methods.51,53, 62,63 3-bromo-7-methoxy-4,5-dihydro-1H-benzo[g]indole-2-carbaldehyde

(5).

7-methoxy-4,5-

dihydro-1H-benzo[g]indole-2-carbaldehyde 6 (255 mg , 1.12 mmol ) was dissolved in a mixture of DMF and CH2Cl2 (1 : 3; 16 mL), and the mixture was cooled to 0 °C. N-bromosuccinimide (228 mg, 1.28 mmol, 1.14 equiv) was added, then the mixture was stirred at 0 °C for 30 min and was quenched with water. The darkened mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The resulting crude mixture was separated by silica gel column chromatography (Petroleum ether : CH2Cl2 = 1 : 3) to afford 5 as a pale green solid (336 mg, 98%). 1H NMR (400 MHz, CDCl3): δ 10.56 (s, 1 H), 9.52 (s, 1 H), 7.63 (d, 1 H, J = 9.2 Hz), 6.85 (m, 2 H), 3.84 (s, 3 H), 2.97 (t, 2 H, J = 7.6 Hz), 2.73 (t, 2 H, J = 7.4 Hz). HRMS-MALDI: [M]+ calcd for C14H13NO2Br+1: 306.0124; found: 306.0124. 1-Br BODIPY (3). A solution of brominated pyrrole carbaldehyde 5 (91.8 mg, 0.3 mmol) and 7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]indole 4 in CH2Cl2 (40 mL) at 0 °C was added POCl3 (34 µL, 0.36 mmol, 1.2 equiv). The resulting mixture was warmed to room temperature slowly and stirred for 12 h. The mixture was cooled to 0 °C and Et3N (0.63 mL, 4.5 mmol, 15 equiv) was added dropwise over 5 min. After stirring for 15 min, BF3•Et2O (0.76 mL, 6 mmol, 20 equiv) was added dropwise to the solution over 5 min. The reaction mixture was warmed to room temperature and stirred for 12 h. The organic layer was washed with water, brine and dried over anhydrous Na2SO4. The solvents were removed under reduced pressure and the residue was purified by flash chromatography (Petroleum ether : CH2Cl2 = 1 : 1) to give the pure product 3 as


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a golden solid (129 mg, 70%). 1H NMR (400 MHz, CDCl3): δ 8.80 (d, 1 H, J = 8.8 Hz), 8.70 (d, 1 H, J = 8.8 Hz), 7.56 (t, 2 H, J = 7.2 Hz), 7.48 (m, 3 H), 7.06 (s, 1 H), 7.01 (m, 2 H), 6.83 (t, 2 H, J = 2.8 Hz), 3.89 (s, 3 H), 3.88 (s, 3 H), 2.95 (m, 4 H), 2.78 (t, 2 H, J = 7.0 Hz), 2.72 (t, 2 H, J = 7.0 Hz).

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C NMR (100 MHz, CDCl3): 161.23, 160.87, 152.65, 149.67, 143.47, 142.65,

139.32, 132.63, 130.70, 130.52, 130.05, 129.83, 129.75, 128.87, 128.25, 121.15, 120.98, 119.50, 114.99, 114.96, 114.46, 114.35, 112.65, 112.57, 55.40, 30.87, 30.32, 21.36, 21.12. HRMSMALDI: [M]+ calcd for C33H26BN2O2F2Br+1: 609.1280; found: 609.1270. 1-NH2 BODIPY (2). To 100 mL acetonitrile solution of 3 (108 mg, 0.18 mmol), sodium azide (35 mg, 0.54 mmol, 3 equiv) were added. The mixture was stirred in dark at 80 °C overnight, monitored with TLC. The reaction was concentrated under vacuum and the resulting residue was purified by silica gel plates (Petroleum ether : CH2Cl2 = 1 : 3) to get BODIPY 2 as a red solid (36 mg, 37 %). 1H NMR (400 MHz, CDCl3): δ 8.80 (d, 1 H, J = 9.2 Hz), 8.69 (d, 1 H, J = 8.8 Hz), 7.50 (t, 2 H, J = 7.2 Hz), 7.42 (m, 3 H), 6.99 (m, 2 H), 6.86 (s, 1 H), 6.80 (t, 2 H, J = 2.4 Hz), 4.17 (s, 2 H), 3.88 (s, 3 H), 3.87 (s, 3 H), 2.92 (m, 4 H), 2.73 (t, 2 H, J = 7.0 Hz), 2.50 (t, 2 H, J = 7.2 Hz).

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C NMR (100 MHz, DMSO): 161.37, 158.93, 156.64, 151.70, 144.17, 142.10,

141.04, 133.86, 132.14, 130.60, 130.42, 130.24, 130.16, 129.14, 127.85, 127.52, 125.52, 122.71, 120.83, 114.66, 114.56, 114.33, 112.56, 112.18, 108.39, 55.83, 55.55, 30.90, 30.29, 21.08, 18.37. HRMS-MALDI: [M]+ calcd for C33H28BN3O2F2+1: 546.2279; found: 546.2274. Probe 1. Under N2, a solution of 2 (33 mg, 0.06 mmol) and pyridine (24 µL, 0.3 mmol, 5 equiv) in 30 mL of CH2Cl2 was added 2,4-dinitrobenzenesulfonyl chloride (160 mg, 0.6 mmol, 10 equiv) in 5 mL of CH2Cl2 dropwise at 0 °C. The reaction solution was stirred overnight under a nitrogen atmosphere and let it warm up to room temperature gradually, during which change of red to green of mixture appeared. The solvents was washed with 1N HCl and brine, dried over


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anhydrous Na2SO4, filtered and evaporated under vacuum. The resulting residue was purified by silica gel plates (Petroleum ether : CH2Cl2 = 1 : 5) to get probe 1 as a black solid (15 mg, 32%). 1

H NMR (400 MHz, CDCl3): δ 8.76 (d, 1 H, J = 8.8 Hz), 8.64 (d, 1 H, J = 9.2 Hz), 8.25 (dd, 1 H,

J = 8.4, 2.0 Hz), 7.96 (d, 1 H, J = 8.8 Hz), 7.83 (d, 1 H, J = 2.0 Hz), 7.54 (m, 4 H), 7.14 (s, 1 H), 7.12 (s, 1 H), 7.01 (m, 2 H), 6.81 (d, 2 H, J = 2.4 Hz), 6.35 (s, 1 H), 3.89 (s, 3 H), 3.88 (s, 3 H), 2.89 (m, 4 H), 2.72 (m, 4 H).

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C NMR (100 MHz, DMSO): 161.52, 161.12, 151.99, 151.96,

150.52, 149.22, 147.67, 144.18, 143.36, 138.91, 136.39, 134.53, 132.72, 132.18, 131.93, 130.09, 129.71, 129.44, 129.36, 128.96, 127.42, 120.84, 120.52, 120.26, 118.76, 114.78, 113.31, 113.14, 55.90, 55.39, 30.29, 29.96, 20.87, 20.19. HRMS-MALDI: [M]+ calcd for C39H30BN5O8F2S+1: 776.1914; found: 776.1907. Crystal structure determination of compound 2 and probe 1. Red crystals (2) and black crystal (probe 1) were grown upon DCM/Hexane mixture solution at 10 °C. The data collections of 2 and probe 1 were performed on a Mac Science DIP2030 imaging plate diffractometer using mirror monochromated Mo-Kα radiation (λ = 0.71073 Å). The unit cell parameters were determined by separately autoindexing several images in each data set using the DENZO program (MAC Science).64 For each data set, the rotation images were collected in 3° increments with a total rotation of 180° about the Ф axis. The data were processed using SCALEPACK. The structure was solved by direct methods and refined anisotropically by the least-squares procedure implemented in the SHELX-97 program system.65 CCDC 987497 (probe 1) and CCDC 987498 (2)

were

deposited

in

Cambridge

Crystallographic

Data

Centre

(http://www.ccdc.cam.ac.uk/conts/retrieving.html). The crystallographic data can be found in supplementary materials. RESULTS AND DISCUSSION


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Synthesis of probe 1. The synthesis of probe 1 was summarized in Scheme 2. Vilsmeier acylation on pyrrole 7 afforded the pyrrol-2-aldehyde 6 which was subsequently brominated with NBS to provide 3-bromo pyrrol-2-aldehyde 5. Condensation with pyrrole 4 and complexation with BF3 etherate generated 1-Bromo BODIPY 3. SNAr replacement of 3 with sodium azide resulted in 1-amino BODIPY 2 which reacted with 2,4-dinitrobenzene-sulfonyl chloride generated the desired probe 1.

N H

O

Br

1. POCl3, DMF ClCH2CH2Cl

CHO

N H

2. NaOAc, H2O O

NBS DMF, CH2Cl2

6

7

N H

O

CHO

5

Br

H 2N NaN3, CH3CN N

N

N

B

B F F

O

POCl3, CH2Cl2

N

F O

O

NEt3, BF3• Et2O

F

N H

O 4

O 3

2 NO2 O

DNBS, Pypidine DCM, 0 °C

HN

S

O NO2 N

N B F F O

O Probe 1

Scheme 2. Synthesis of Probe 1. Absorption and fluorescence spectra of probe 1 upon addition of analytes. There were distinct changes in the absorption and emission of probe 1 upon addition of thiophenol (PhSH) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) at 37 °C (Fig. 1). The absorption maximum of probe 1 was 640 nm (ε = 1.01 × 105 Μ−1 cm−1). Probe 1 exhibited no fluorescence with λex = 580 nm. Upon addition of PhSH (2 × 10−4 M, 20 equiv), while the absorption at 640 nm


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decreased abruptly, accompanied by an increase in the absorption at 608 nm, fluorescence intensity increased a few seconds later and reached the maximum after about 90 min at 633 nm. These results indicated that the chemical reaction between the thiophenol and the masked sulfonamide moiety interrupted the PET process of probe 1, and 1-NH2 BODIPY 2 (λmax = 633 nm, ε = 8.02 × 104 Μ−1 cm−1) with high fluorescence quantum yield (φ = 0.63) was released.66 The reaction product was monitored and confirmed by comparison studies with standard pure 2 through TLC and 1H NMR. Our results demonstrate that probe 1 is a useful fluorescent probe for detecting thiophenol with red emission which may greatly reduce background absorption, fluorescence, light scattering, and improve the detectable sensitivity67,68. The big off-to-on (φ: 0→0.63) contrast ratio and dramatic change of fluorescence (Fig. 1b, inset) upon addition of PhSH allow sensitive identification of thiophenols with “naked eye”.

Figure 1. The sensitivity of probe 1 was then studied by fluorescence response toward various concentrations of PhSH. Upon incremental addition of PhSH with concentration range of 0.1 µM-1 mM in probe 1 solution (10 µM) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) at


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37 °C for 60 min, absorption band centered at 640 nm gradually decreased, accompanied by increase of absorption at 608 nm (isosbestic point at 621 nm), and a concurrently increased fluorescence at 633 nm was observed (Fig. 2a and Fig. 2b). Further study suggested a concentration-dependent response manner (Fig. 2c) with the linear response of probe 1 toward PhSH ranging from 6 × 10−6 M to 1 × 10−4 M (Fig. 2c inset).

Figure 2. To investigate the selectivity, probe 1 (10 µM) was treated with thiophenols, aliphatic thiols, natural amino acids and other common nucleophiles in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) at 37 °C, and absorption/fluorescence spectra were followed (Fig. S1 and S2). As shown in Fig. S1, only thiophenols induced significant blue shift in the absorption spectra and various individual thiophenol responded very similarly. The luminescence intensity was significantly enhanced at 633 nm in the presence of thiophenols (200 µM) such as 3-aminothiophenol, 2aminobenzenethiol, and p-toluenethiol, 4-chlorothiophenol, as well as thiophenol (Fig. S2). No noticeable changes in the absorption and emission spectra were observed upon addition of other analytes (400 µM) including NaN3, KI, D-alanine, PhOH, PhNH2, glycine, GSH, Hcy, Cys, CH3(CH2)7SH, and CH3CH2SH. The sensitive discrimination of aromatic thiols allowed a visual distinction of thiophenols from other analytes with naked eye under ambient light or under UV irradiation (Fig. S3).


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Competition experiments were carried out by adding PhSH (200 µM) to the solution of probe 1 in the presence of 400 µM of aliphatic thiols, natural amino acids and other common nucleophiles (Fig. 3). Strong fluorescence was visualized when probe 1 exposed to PhSH, which was not disturbed by non-thiophenol type analytes. This result indicates that probe 1 exhibited high selectivity toward thiophenols.

Figure 3. The effect of pH on the fluorescence intensity and reactivity of probe 1 (10 µM) was examined in the absence and presence of thiophenol in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM) at 37 °C for 60 min (Fig. S4). Probe 1 is stable in pH range of 1.0–13.0. However, the sulfonamide moiety was easily removed by highly nucleophilic thiolate anion through SNAr process, concurrently, a remarkable enhancement of the fluorescence was observed in the presence of PhSH in a wide pH range (3.0–13.0) and the preferred pH range was 5.0-8.0. The weaker response at pH > 8.0 was presumably attributed to diminished concentration of thiophenol due to the disulfide formation under basic condition.


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Application of probe 1 was demonstrated by the live cell fluorescence microscopic technique (Figures 4 and S6). After incubated with probe 1 in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3) at 37 °C for 30 min, Human Hepatoma SMMC-7721 cell showed non-detectable fluorescence by fluorescence imaging (Fig. S6), in a stark contrast, strong fluorescence in the cell cytoplasm was identified once incubated with PhSH for another 30 min. The imaging of thiophenol in SMMC-7721 could be easily visualized even when 1 µM of thiophenol was used for incubation for 30 min (Fig. 4). These results demonstrate the usefulness of probe 1 in the selective detection of thiophenols in the intracellular environment by discriminating small molecule biothiols.

Figure 4. Probe 1 also sensitively responded to the presence of thiophenol in PBS buffer containing 1% Tween (Fig. S5). To validate the practicality, probe 1 was employed to determine thiophenol in water. 1% Tween 20 was used to assist probe 1 dissolving in water. Upon addition of PhSH (18.5 µM, 18.5 equiv), fluorescence intensity increase was observed in a few seconds and reached the maximum within 30 min at 632 nm. The sensitivity of probe 1 (10 µM) was then studied by fluorescence response toward various concentrations of PhSH in acetonitrile (minimum amount).


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The response profile of probe 1 towards various concentration of PhSH in water (containing 1% Tween 20) was shown in Fig. 5. Linear response to very low concentration of PhSH was observed. The detection limit for PhSH was determined to be 37 nM which corresponds to 4 ppb.

Figure 5. For comparison purposes, a comprehensive summary of the recently developed fluorescent probes for thiophenols is collected in Table S1. The probe 2 showed the longest wavelength for the response and exhibited excellent analytical performance. The faster response rate and higher sensitivity achieved in water containing 1% Tween 20 provided a convenient discrimination method for aromatic thiols. Such high selectivity could be visualized by the photo image shown in Fig. 6. Probe 1 (10 µM) was treated with PhSH (18.5 µM) in comparison with excessive aliphatic thiols, natural amino acids, other common nucleophiles in water with 1% Tween 20. Only PhSH resulted strong red fluorescence, which clearly demonstrates the fast and sensitive discrimination of thiophenol.


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Figure 6.

Figure captions Figure 1. Absorption and fluorescence emission (λex = 580 nm) time profile of probe 1 (10 µM) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3, 37 °C) with PhSH (200 µM) after the specified time periods (0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 and 100 min). Figure 2. UV-vis absorption (a) and fluorescence spectra (b, λex = 580 nm) of the probe 1 (10 µM) upon addition of increasing concentrations of PhSH (0.01−100 equiv) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3, 37 °C) for 60 min. Concentration dependent fluorescence intensity changes of the probe 1 (10 µM) were monitored at 633nm. The inner panel displays the linear relationship of fluorescence enhancement of probe 1 toward PhSH from 6 × 10−6 M to 1 × 10−4 M and the linear relationship is y = 4.341 + 0.604x (R = 0.9998). Figure 3. The selectivity of probe 1 (10 µM) toward thiophenols (200 µM ) and other substances (400 µM) in acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.3, 37 °C) for 100 min. Black bar: the fluorescence intensity of only a single analyte with the probe 1. Red bar: the fluorescence intensity of a mixture of analyte and PhSH with the probe 1 (λex = 580 nm and λem = 633 nm). (1) probe 1 only, (2) NaN3, (3) KI, (4) D-Alanine, (5) PhOH, (6) PhNH2, (7) Glycine,


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(8) GSH, (9) Hcy, (10) Cys, (11) CH3(CH2)7SH, (12) CH3CH2SH, (13) 3-Aminothiophenol, (14) 2-Aminobenzenethiol, (15) p-Toluenethiol, (16) 4-Chlorothiophenol, (17) PhSH. Figure 4. Photographs of thiophenol in living cells with living 7721 cells. Bright field (a) and fluorescence (b) images of 7721 cells incubated with thiophenol (1 µM) for 30 min after pretreatment with probe 1 (10 µM) for 30 min. Figure 5. Fluorescence response of probe 1 (10 µM) in water (containing 1% Tween 20) at 37 °C with PhSH with λex = 580 nm and λem = 632 nm. (a) Time course response upon addition of PhSH (18.5 µM); (b) Concentration-dependent response upon addition of PhSH recorded after 25 min. The inner panel displays the fluorescence enhancement of probe 1 toward PhSH (185 nM−27 µM) and the linear relationship is y = 1.54 + 2.144x (R = 0.9997).

Figure 6. Photographs of probe 1 (10 µM) with thiophenol (18.5 µM), aliphatic thiols, natural amino acids and other common nucleophiles (400 µM) in Tween 20 water solution for 10 min at 10 °C taken under ambient light and under a hand-held UV lamp with λex = 365 nm. (1) probe 1 only, (2) NaN3, (3) KI, (4) D-Alanine, (5) PhOH, (6) PhNH2, (7) Glycine, (8) GSH, (9) Hcys, (10) Cys, (11) CH3(CH2)7SH, (12) CH3CH2SH, (13) PhSH.


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CONCLUSION In conclusion, we have developed a novel 1-amino substituted BODIPY-based fluorescent probe 1 with high selectivity and sensitivity for thiophenols. Probe 1 is promising for the quantitative detection of thiophenol with linear response from 6 × 10−6 M to 1 × 10−4 M and the detection limit for PhSH reaches 4 × 10−6 M measured in acetonitrile/PBS buffer. Moreover, probe 1 is a useful fluorescent probe for detecting thiophenols in living cells in red emission which may greatly improve the detectable sensitivity. The detection of thiophenols was able to be conducted in water when 1% Tween 20 was used and the sensitivity was further improved with the detection limit to 4 ppb.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by NSFC (21372063) and Program for Changjiang Scholars and Innovative Research Team in University, No. PCS IRT1126. Supporting Information. Selected crystallographic data for compound 1 and 2, absorption and emission spectra and photo images in response to various analytes, pH response, comparison of fluorescent probes for thiophenol, cell imaging, as well as copies of NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.


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REFERENCES (1) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019-6031. (2) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351-5358. (3) Herzenberg, L. A.; De Rosa, S. C.; Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.; Deresinski, S. C.; Herzenberg, L. A. Proc. Natl. Acad. Sci. USA. 1997, 94, 1967-1972. (4) Heafield, M. T.; Fearn, S.; Steventon, G. B.; Waring, R. H.; Williams, A. C.; Sturman, S. G. Neurosci. Lett. 1990, 110, 216-220. (5) Nygard, O.; Nordrehaug, J. E.; Refsum, H.; Ueland, P. M.; Farstad, M.; Vollset, S. E. N. Engl. J. Med. 1997, 337, 230-236. (6) Jacobsen, D. W. Clin. Chem. 1998, 44, 1833-1843. (7) Sofia, C.; Chadefaux, B.; Coude, M.; Oaillard, O.; Kamoun, E. Clin. Chem. 1990, 36, 2137-2138. (8) Townsend, D. M.; Tew, K. D.; Tapiero, H. Biomed. Pharmacother. 2003, 57, 145-155. (9) Roy, K.-M. Thiols and Organic Sulfides, in Ullmann’s Encyclopedia of Industrial Chemistry, John Wiley & Sons, New York, 7th ed, 2007. (10) Shimada, K.; Mitamura, K. J. Chromatogr., B: Biomed. Sci. Appl. 1994, 659, 227-241. (11) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103-1170.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

(12) Eychmuller, A.; Rogach, A. L. Pure Appl. Chem. 2000, 72, 179-188. (13) Juneja, T. R.; Gupta, R. L.; Samanta, S. Toxicol. Lett. 1984, 21, 185-189. (14) Amrolia, P.; Sullivan, S. G.; Stern, A.; Munday, R. J. Appl. Toxicol. 1989, 9, 113-118. (15) Munday, R. J. Appl. Toxicol. 1985, 5, 402-408. (16)

Material

safety

data

sheet

of

thiophenol

from

Sigma-Aldrich,

http://www.castleviewuk.com/Frameless/Safe/msds/ex/MSDS_thiophenol.htm. (17) Hathaway, G. J.; Proctor, N. H. Proctor and Hughes’ Chemical Hazards of the Workplace, John Wiley & Sons, Inc, New Jersey, 2004, 575-576. (18) Tang, Y.; Yang, H. R.; Sun, H. B.; Liu, S. J.; Wang, J. X.; Zhao, Q.; Liu, X. M.; Xu, W. J.; Li, S. B.; Huang, W. Chem.-Eur. J. 2013, 19, 1311-1319. (19) Li, X.; Qian, S.; He, Q.; Yang, B.; Li, J.; Hu, Y. Org. Biomol. Chem. 2010, 8, 3627-3630. (20) Sun, W.; Li, W.; Li, J.; Zhang, J.; Du, L.; Li, M. Tetrahedro. 2012, 68, 5363-5367. (21) Sun, W.; Li, W.; Li, J.; Zhang, J.; Du, L.; Li, M. Tetrahedron Lett. 2012, 53, 2332-2335. (22) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922–2925. (23) Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8597-8600. (24) Jiang, W.; Cao, Y.; Liu, Y.; Wang, W. Chem. Commun. 2010, 46, 1944-1946. (25) Lin, W.; Long, L.; Tan, W. Chem. Commun. 2010, 46, 1503-1505.


20

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Zhao, C.; Zhou, Y.; Lin, Q.; Zhu, L.; Feng, P.; Zhang, Y.; Cao, J. J. Phys. Chem. B 2011, 115, 642-647. (27) Zhao, W.; Liu, W.; Ge, J.; Wu, J.; Zhang, W.; Meng, X.; Wang, P. J. Mater. Chem. 2011, 21, 13561-13568. (28) Kand, D.; Mishra, P. K.; Saha, T.; Lahiri, M.; Talukdar, P. Analyst 2012, 137, 3921-3924. (29) Zhang, R.; Ye, Z.; Yin, Y.; Wang, G.; Jin, D.; Yuan, J.; Piper, J. A. Bioconj. Chem. 2012, 23, 725-733. (30) Wang, X.; Cao, J.; Zhao, C. Org. Biomol. Chem. 2012, 10, 4689-4691. (31) Wang, Z.; Han, D. M.; Jia, W. P.; Zhou, Q. Z.; Deng, W. P. Anal. Chem. 2012, 84, 49154920. (32) Liu, X. L.; Duan, X. Y.; Du, X. J.; Song, Q. H. Chem.-Asian J. 2012, 7, 2696-2702. (33) Zhao, C.; Wang, X.; Cao, J.; Feng, P.; Zhang, J.; Zhang, Y.; Yang, Y.; Yang, Z. Dyes Pigm. 2013, 96, 328-332. (34) Li, J; Zhang, C.-F; Yang, S.-H; Yang, W.-C; Yang, G.-F. Anal. Chem. 2014, 85, 30373042. (35) Kand, D.; Mandal, P. S.; Datar, A.; Talukdar, P. Dyes Pigm. 2014, 106, 25-31. (36) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891-4932. (37) Michel, B. W.; Lippert, A. R.; Chang, C. J. J. Am. Chem. Soc. 2012, 134, 15668-15671. (38) Lee, C. H.; Yoon, H. J.; Shim, J. S.; Jang, W. D. Chem.-Eur. J. 2012, 18, 4513-4516.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

(39) Hu, R.; Gomez-Duran, C. F. A.; Lam, J. W. Y.; Belmonte-Vazquez, J. L.; Deng, C.; Chen, S.; Ye, R.; Pena-Cabrera, E.; Zhong, Y.; Wong, K. S.; Tang, B. Z. Chem. Commun. 2012, 48, 10099-10101. (40) Burghart, A.; Thoresen, L. H.; Chen, J.; Burgess, K.; Bergström, F.; Johansson, L. B. Å. Chem. Commun. 2000, 2203-2204. (41) Beer, G.; Niederalt, C.; Grimme, S.; Daub, J. Angew. Chem., Int. Ed. 2000, 39, 32523254. (42) Ziessel, R.; Ulrich, G.; Olivier, J. H.; Bura, T.; Sutter, A. Chem. Commun. 2010, 46, 79787980. (43) Bochkov, A. Y.; Akchurin, I. O.; Dyachenko, O. A.; Traven, V. F. Chem. Commun. 2013, 49, 11653-11655. (44) Buyukcakir, O.; Bozdemir, O. A.; Kolemen, S.; Erbas, S.; Akkaya, E. U. Org. Lett. 2009, 11, 4644-4647. (45) Jiao, C.; Zhu, L.; Wu, J. Chem.-Eur. J. 2011, 17, 6610-6614. (46) Umezawa, K.; Nakamura, Y.; Makino, H.; Citterio, D.; Suzuki, K. J. Am. Chem. Soc. 2008, 130, 1550-1551. (47) Jiao, C.; Huang, K.-W.; Wu, J. Org. Lett. 2011, 13, 632-635. (48) Jokic, T.; Borisov, S. M.; Saf, R.; Nielsen, D. A.; Kühl, M.; Klimant, I. Anal. Chem. 2012, 84, 6723-6730. (49) Adarsh, N.; Shanmugasundaram, M.; Ramaiah, D. Anal. Chem. 2013, 85, 10008-10012.


22

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(50) Zhao, W.; Carreira, E. M. Angew. Chem., Int. Ed. 2005, 44, 1677-1679. (51) Zhao, W.; Carreira, E. M. Chem.-Eur. J. 2006, 12, 7254-7263. (52) Jiang, X. -D.; Zhang, J.; Shao, X.; Zhao, W. Org. Biomol. Chem. 2012, 10, 1966-1968. (53) Jiang, X. -D.; Gao, R.; Yue, Y.; Sun, G. T.; Zhao, W. Org. Biomol. Chem. 2012, 10, 68616865. (54) Jiang, X. -D.; Zhang, H.; Zhang, Y.; Zhao, W. Tetrahedron 2012, 68, 9795-9801. (55) Emrullahoğlu, M.; Üçüncü, M.; Karakuşa, E. Chem. Commun. 2013, 49, 7836-7838. (56) Park, J.; Kim, H.; Choi, Y.; Kim, Y. Analyst 2013, 138, 3368-3371. (57) Boens, N.; Leen, V.; Dehaen, W. Chem. Soc. Rev. 2012, 41, 1130-1172. (58) Gawley, R. E.; Mao, H.; Haque, M. M.; Thorne, J. B.; Pharr, J. S. J. Org. Chem. 2007, 72, 2187-2191. (59) Coskun, A.; Yilmaz, M. D.; Akkaya, E. U. Org. Lett. 2007, 9, 607-609. (60) Hall, M. J.; Allen, L. T.; O'Shea, D. F. Org. Biomol. Chem. 2006, 4, 776-780. (61) Fukuyama, T.; Cheung, M.; Jow, C-K.; Hidai, Y.; Kan, T. Tetrahedron Lett. 1997, 38, 5831-5834. (62) Schmidt, E. Y.; Mikhaleva, A. I.; Vasil’tsov, A. M.; Zaitsev, A. B.; Zorina, N. V. Issue in Honor of Prof. Vladimir I. Minkin. ARKAT USA, Inc. 2005, 11-17. (63) Kang, D. J.; Eom, D.; Mo, J.; Kim, H.; Sokkalingam, P.; Lee, C-H.; Lee, P. H. Bull. Korean Chem. Soc. 2010, 31, 507-510.


23


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Page 24 of 25

(64) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326. (65) Sheldrick,G. M. SHELXS97 and SHELXL97, University of Gottingen, Germany, 1997. (66) Quantum yield was determined by using 1,7-diphenyl-3,5-di(p-methoxyphenyl)-azaBODIPY, namely BF2 chelate of [5-(4-methoxyphenyl)-3-phenyl-1H-pyrrol-2-yl]-[5-(4methoxylphenyl)-3-phenylpyrol-2-ylidene]amine (Ф = 0.36) as reference, see: Gorman, A.; Killoran, J.; O’Shea, C.; Kenna, T.; Gallaghter, W. M.; O’Shea, D. F. J. Am. Chem. Soc. 2004, 126, 10619-10631. (67) Ntziachristos, V.; Ripoll, J.; Weissleder, R. Opt. Lett. 2002, 27, 333-335. (68) Sowell, J.; Strekowski, L.; Patonay, G. J. Biomed. Opt. 2002, 7, 571-575.


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Highly Selective and Sensitive 1-Amino BODIPY-Based Red

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