A solvent depend on ratiometric fluorescent probe for hypochlorous acid and its application in living cells

Article abstract of DOI:10.1016/j.dyepig.2016.09.048

Hypochlorous acid (HOCl)/hypochlorite (OCl^?) plays an important role in immune system, while as a major reactive oxygen species (ROS) in living systems, HOCl/OCl^? is responsible for some human diseases. Thus it is critical to monitor the change of HOCl in the living cells. In this study, we prepared a novel fluorescent probe BDP-OX based on BODIPY-Dialdoxime for HOCl/OCl^? determination, which displayed a remarkable “ratio” fluorescence response towards HOCl/OCl^? with a fast response (completed within 15?s). The ratios of emission intensities at 538–589?nm (I₅₃₈/I₅₈₉) displayed a large increase from 0.015 to 2.88 with this probe. More importantly, confocal fluorescence imaging with BDP-OX showed separated signals in the presence of HOCl/OCl^? in living A549?cells, that indicating BDP-OX have potential biological application prospects.

Full text of DOI:10.1016/j.dyepig.2016.09.048

Accepted Manuscript
A solvent depend on ratiometric fluorescent probe for hypochlorous acid and its
application in living cells
Jin Kang, Fangjun Huo, Yongkang Yue, Ying Wen, Jianbin Chao, Yongbin Zhang,
Caixia Yin
PII:
S0143-7208(16)30675-1
10.1016/j.dyepig.2016.09.048
DYPI 5500
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 7 September 2016
Revised Date: 20 September 2016
Accepted Date: 20 September 2016
Please cite this article as: Kang J, Huo F, Yue Y, Wen Y, Chao J, Zhang Y, Yin C, A solvent depend on
ratiometric fluorescent probe for hypochlorous acid and its application in living cells, Dyes and Pigments
(2016), doi: 10.1016/j.dyepig.2016.09.048.
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Abstract Graphic
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The Title:
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A solvent depend on ratiometric fluorescent probe for hypochlorous
acid and its application in living cells
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List of Authors:
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Jin Kang,a Fangjun Huo,b Yongkang Yue,a Ying Wen,a Jianbin Chao,b Yongbin Zhangb and Caixia Yin*a
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A fluorescent sensor based on BODIPY fluorophore integrated with two
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aldoxime moiety, displayed a remarkable fluorescence “ratiometric”
response to hypochlorous acid (HOCl) with low detection limits and high
selectivity. The oxidative dehydrogenation of BODIPY dialdoxime by
HOCl results in a distinct fluorescence maximum emission change from
589 nm to 538 nm accompany with fluorescence color change from orange
to green.
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21 A solvent depend on ratiometric fluorescent probe for hypochlorous
22 acid and its application in living cells
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Jin Kang,a Fangjun Huo,b Yongkang Yue,a Ying Wen,a Jianbin Chao,b Yongbin Zhangb and Caixia Yin*a
a
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Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi
Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China.
b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China.
*Corresponding author: C. X. Yin, E-mail: yincx@sxu.edu.cn, Tel/Fax:
+86-351-7011022.
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Abstract: Hypochlorous acid (HOCl)/hypochlorite (OCl-) plays an important role in
immune system, while as a major reactive oxygen species（ROS）in living systems,
HOCl/OCl- is responsible for some human diseases. Thus it is crutical to monitor the
change of HOCl in the living cells. In this study, we prepared a novel fluorescent probe
BDP-OX based on BODIPY-Dialdoxime for HOCl/OCl- determination, which
displayed a remarkable “ratio” fluorescence response towards HOCl/OCl- with a fast
response (completed within 15 s). The ratios of emission intensities at 538 to 589 nm
(I538/I589) displayed a large increase from 0.015 to 2.88 with this probe. More
importantly, confocal fluorescence imaging with BDP-OX showed separated signals in
the presence of HOCl/OCl- in living A549 cells, that indicating BDP-OX have potential
biological application prosepects.
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Keywords: Ratiometric; HOCl/OCl-; Solvent depend; Fluorescent probe; BODIPY
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1. Introduction
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Hypochlorous acid (HOCl), one of the vital reactive oxygen species (ROS) in
biological organisms, plays important roles in diverse normal biochemical functions and
abnormal pathological processes [1-3]. Ogenous hypochlorous acid is produced mainly
in leukocytes (including neutrophils, macrophages, and monocytes) by myeloperoxidase
(MPO)-catalyzed peroxidation of chloride ions [4]. On the one hand, the moderate
hypochlorous acid act as a kind of effective antimicrobial agent for the immune system,
which plays a key role in killing a wide range of pathogens [5]. On the other hand,
abnormal production of HOCl had resulted in a variety of diseases, including hepatic
ischemia-reperfusion injury [6], atherosclerosis [7], lung injury [8], rheumatoid arthritis
[9], and certain cancers [10]. Meanwhile, HOCl could partially dissociate to
hypochlorite (OCl-) in aqueous solutions at physiological pH, which often used as a
disinfectant and excessive hypochlorite could also exert detrimental effect on tissues
[11]. Therefore, it has become more significant to monitor the cellular HOCl
concentration for biological research and clinical diagnosed.
As we all know, HOCl is highly reactive but short-lived under the physiological
conditions [12] and the average level of HOCl generation is relatively low in living cell
about 0.47 nmol/min per 106 neutrophils cells [13]. Therefore, looking for a desirable
method with fast response and high sensitivity is necessary for the detection of HOCl,
which is conductive to the real-time monitoring of the fluctuation of HOCl content in its
cellular site of action [14]. A majority of methods for detection of HOCl have been
developed [15-20]. Among them, fluorescence probes are generally superior in terms of
sensitivity, temporal-spatial resolution, and ease of use. Although a lot of HOCl probes
have been fabricated [21-29], most of them respond to HOCl only with changes in
fluorescent intensity, while the researches about the ratiometric HOCl probes are
relatively fewer. A major limitation of intensity-based probes is that the quantitative
measurements might be influenced by the problematic variations in the probe
distribution and sample environment [30]. By contrast, ratiometric fluorescent probes
provide a built-in correction for environmental effects, which can also increase the
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dynamic range of fluorescence measurement and allow measuring the emission
intensities at two different wavelengths [31]. Therefore, we intend to design a
fluorescent probe with a ratiometric response to HOCl.
Here, we have designed a novel ratiometric fluorescent probe by integrating the
BODIPY (boron-dipyrromethene) fluorescent with double aldoxime units for selective
detection of HOCl (Scheme 1). This probe employed the BODIPY fluorophore as the
signal reporter owing to its excellent photophysical properties such as long emission,
and high molar absorption coefficients and fluorescence quantum yield [32-36]. As a
representive and sensitive functional group for the identification of hypochlorous acid,
the oxime has been well established in the fluorescent probes, aiming at provideing a
selective signalling platform for the detection of HClO [37-42].
<Inserted Scheme 1>
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2. Materials and Methods
2.1. Materials
Sodium hydroxide solution (0.1 M) was added to aqueous PBS (10 mM) to adjust the
pH to 7.4. Sodium hypochlorite (NaClO) was prepared by diluting 14.5% aqueous
solution. Reagents with analytical grades and demineralized water were used for
preparing the solutions. Stock solutions (2 mmol/L) of CN-, F-, Cl-, Br-, NO3 , NO2
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SO3 , SCN-, AcO-, CO3 , SO4 , ClO2 , ClO3 , ClO4 , HS-, CO3 , PO4 ，were prepared
by direct dissolution of proper amounts of sodium salts. All other chemicals used were
of analytical grade.
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3-
2.2. Instruments
A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. TLC analysis
was performed using precoated silica plates. HITACHI F-7000 fluorescence
spectrophotometer was employed to measure fluorescence spectra. Shanghai Huamei
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Experiment Instrument Plants, China, provided a PO-120 quartz cuvette (10 mm). H
NMR and 13C NMR experiments were performed with a Bruker AVANCE-600 MHz
and 150 MHZ NMR spectrometer, respectively (Bruker, Billerica, MA). Coupling
constants (J values) are reported in hertz. ESI determinations were carried out on AB
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Triple TOF 5600plus System (AB SCIEX, Framingham, USA). The crystal data was
determined by a D8 Venture X-ray crystallography. The ability of probe reacting to
hypochlorite in the living cells was also evaluated by Zeiss LSM880 Airyscan confocal
laser scanning microscope.
2.3 Cell culture
A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) in
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an atmosphere of 5% CO2 and 95% air at 37 C. CLSM Imaging. Cells were plated on
14 mm glass coverslips and were incubated overnight. The cells were washed with PBS
and then incubated with 2 µM BDP-OX in dimethyl sulfoxide (DMSO)/PBS (0.5%, v/v)
for 3 h at 37 °C. After washing three times, the cells were subjected to CLSM imaging
with the excitation wavelength at 488 nm. The green and red channels were set at 525 ±
25 and 595 ± 25 nm, respectively. A549 cells stained with 2 µM BDP-OX for 30 min at
37 °C showed pink fluorescence. In a further experiment that A549 cells displayed a
green fluorescence when the cells were first incubated with 2 µM of probe for 30 min at
37 °C and then incubated with NaClO (10 µM), that were excited at 488 nm, and the
emission was collected at 525 ± 25 nm.
2.4. Preparation and characterization of probes
2.4.1Preparation and characterization of compound 1
The benzaldehyde (0.424g, 4 mmol) and 2,4-dimethylpyrrole (0.833 g, 8.7 mmol)
were dissolved in dry CH2Cl2 (200 mL), trifluoroacetic acid (TFA) were added to the
reaction mixture, the mixture was stirred for 12 h. DDQ (0.98 g, 4 mmol) was added to
the reaction mixture. 9 mL of triethylamine and 9 mL of BF3·OEt2 were added to the
mixture. crude product was purified by column chromatography to obtain orange-red
crystal solid 1 (0.38 g, 30%). 1H NMR (600 MHz, DMSO-d6): δ 7.57 (d, J =6.0 Hz, 3H),
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7.38 (d, J = 6 Hz, 2H), 6.18 (s, 2H), 2.45 (s, 6H), 1.33 (s, 6H). C NMR (150 MHz,
DMSO-d6): δ 155.31, 143.19, 142.41, 134.45, 131.14, 129.75, 128.18, 121.85, 14.69,
14.40. ESI-MS m/z: [M + H]+ calcd for 324.16; Found 325.16.
2.4.2 Preparation and characterization of compound 2
A mixture of DMF (6 mL) and POCl3 (6 mL) was stirred, to this reaction mixture was
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added compound 1 (149 mg, 0.42 mmol) in dichloroethane (20 mL), and the mixture
was stirred for 2 h. The crude product was further purified using column
chromatography (silica gel, hexane/EtOAc/CH2Cl2, 4/1/1, v/v) to give BODIPY 2 (142
mg, 89%) as red crystal solid.1H NMR (600 MHz, DMSO-d6): δ 9.95 (s, 1H), 7.60 (s,
3H), 7.43 (s, 2H), 6.45 (s, 1H), 2.71 (s, 3H), 2.55 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H). 13C
NMR (150 MHz, DMSO-d6): δ 187.09, 162.16, 155.56, 147.61, 144.10, 142.26, 133.79,
130.16, 129.97, 129.43, 128.13, 126.38, 124.91, 15.21, 13.11, 11.59. ESI-MS m/z: [M +
H]+ calcd for 352.19; Found 352.92.[M+Na]+ Calcd for 374.18; Found 374.83.
2.4.3 Preparation and characterization of compound 3
A mixture of DMF (6 mL) and POCl3 (6 mL) was stirred, to this reaction mixture was
added compound 2 (160 mg, 0.42 mmol) in dichloroethane (20 mL), and the mixture
was stirred for 5 h. The crude product was further purified using column
chromatography (silica gel, hexane/EtOAc/CH2Cl2, 4/1/1, v/v) to give BODIPY 3 (141
mg, 82%) as red crystal solid. 1H NMR (600 MHz, CDCl3): δ 10.06(s, 2H), 7.60 (s, 3H),
7.31 (s, 2H), 2.89 (s, 6H), 1.71 (s, 6H). 13C NMR (150 MHz, CDCl3): δ 185.55, 160.75,
148.39, 147.26, 133.58, 133.55, 131.87, 130.18, 130.16, 129.91, 129.88, 128.10, 127.39,
127.36, 13.80, 12.03. ESI-MS m/z: [M + H]+ calcd for 353.19; Found 352.92. [M +
Na]+ Calcd for 375.18; Found 374.83.
2.4.4 Preparation and characterization of BDP-OX
Compound 3 (114 mg, 0.3 mmol) and hydroxylamine hydrochloride (41.4 mg, 0.6
mmol) in absolute ethanol (10.0 mL) were stirred at reflux temperature for 1 hour, the
residue was purified by column chromatography (hexane/ethyl acetate (4/1)).
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BODIPY-dioxime was obtained as red solid (86.0 mg, 70.0% yield). H NMR (600
MHz, CDCl3) δ: 11.17(s, 2H), 8.08(s, 2H), 7.60(s, 3H), 7.42(s, 2H), 2.64(s, 6H), 1.43(s,
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6H). C NMR (150 MHz, CDCl3): δ 167.42, 155.65, 143.51, 142.85, 134.26, 132.16,
131.99, 131.07, 130.07, 129.93, 129.13, 128.34, 123.98, 30.47, 19.12, 14.48, 12.65.
ESI-MS m/z: [M + H ]+ calcd for 381.20; Found 380.92. Crystal size: 0.200 x
0.160 x 0.090 mm, monoclinic, space group P 2(1)/c. a=9.1318 (6) Å, b= 13.7392(9) Å,
c=16.5704(11) Å, α= 90 o, β=103.270(2) o, γ= 90 o. V =2023.5(2) Å3，Z= 4, T=300(2) K,
o
θmax=28.298 . 19891 refections measured, 5007 unique (Rint = 0.0511). Final residual
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for 277 parameters and 5007 refections with I > 2σ (I): R1 = 0.0599, wR2 = 0.01361 and
GOF = 1.018 (Fig. 1 and S5, ESI†).
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<Inserted Figure 1>
2.5. General UV-Vis and fluorescence spectra measurements
Stock solutions of probes (2 mM) were prepared in CH3OH. Stock solutions (2.0 mM)
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of various analytes including ClO−, H2O2, ClO2 , ONOO−, F−, ClO3 , CN−, NO2 , S2−,
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SCN−, MnO4 , ClO4 , CO3 and P2O7 were prepared in deionized water. All UV/Vis
and fluorescence titration experiments were performed using PBS (10 mM, pH 7.0)
solution with at room temperature. And any changes in the fluorescence intensity were
monitored using a fluorescence spectrometer (λex = 488 nm, slit: 5 nm/5 nm).
2.6
．The detection limit
The detection limit was found to be 0.85 µM from 10 blank solutions based on the
definition by IUPAC (CDL= 3Sb/m) [43]. Detection limit for HOCl was calculated by
Eq. (1): Detection limit = 3σ/m (1). Where σ = standard deviation of 10 blank
measurements and m = slope obtained from the graph of fluorescence intensity ratio vs
concentration of HOCl [44]. BDP-OX is able to detect low micromolar concentrations
of hypochlorite.
3. Results and discussion
3.1. The Selective response of probe to hypochlorite
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BDP-OX was treated with various relevant analyte (100 equiv.) including CN , F , Cl ,
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Br , NO3 , NO2
, SO3 , SCN , AcO , CO3 , SO4 , ClO2 , ClO3 , ClO4 , HS , CO3 ,
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PO4 , Cys，Hcy, GSH, in PBS : DMSO = 1:9 (V/V pH = 7.4), then their fluorescence
emission was determinated by fluorescence spectrophotometer. As shown in Fig. 2, a
significant “ratiometric” response was observed with OCl-, while other analytical
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samples exhibited no conspicious increasement in the fluorescene response.
Corresponding fluorescent color changes were shown under illumination with a 365 nm
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UV lamp (Fig. S4). The outstanding selectivity for OCl- over other analytes indicates
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the BDP-OX can be applied into OCl- detection in complex biological environments or
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living organism.
<Inserted Figure 2>
For the selective recognition of OCl− in practical samples, it is crucial to investigate
the OCl− signaling of BDP-OX in the presence of common metal ions or anions as
background. With the tested metal ions, the ratios of emission intensities at 538 and 589
nm (I538/I589) varied in a narrow range (Fig. 3). These observations clearly imply that
BDP-OX could be applied for the detection of OCl- in chemical and environmental
analytes, where various metal ions and anions are frequently present as coexisting ions.
<Inserted Figure 3>
3.2. UV-is and fluorescence spectra of detecting hypochlorite.
The basic spectroscopic properties of BDP-OX were assessed in solution PBS : DMSO
= 1:9 (V/V pH = 7.4). As the absorption spectra (Fig. 4) depicted, the BDP-OX
exhibited a maximum absorption at 535 nm and the maximum absorption peak
presented an obvious decreasing and a blue-shift after the addition of OCl-. The
weakening of maximum absorption intensity can be attributed to the reduced
concentration of BDP-OX. The hypochromatic shift of the absorption can attribute to
the ICT from the BODIPY fluorophore to the oxime group.
<Inserted Figure 4>
The fluorescence spectra measurements were conducted by titrating the
aqueous solution of OCl-（2×10-3）gradually into PBS : DMSO = 1 : 9 (V/V pH = 7.4)
solution containing 10.0 µM probe (with a pink fluorescence), at the same time, any
changes in the fluorescence intensity were monitored using a fluorescence spectrometer
(λex = 488 nm, slit: 5 nm/5 nm). The fluorescent intensity of BDP-OX exhibited a series
of remarkable changes with the increasing concentrations of OCl- ions. The intensity of
the emission maximum at 589 nm decreased and blue-shift gradually accompany with
the appearance of a new blue-shifted emission band centered at 538 nm (Figure 5).
Importantly, the ratios of emission intensities at 538 and 589 nm (I538/I589) exhibiting a
large increase from 0.015 to 2.88, which could be attribute to the rapid isomerization of
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the “C=N-OH” group. And the ratios of emission intensities at the wavelength of 538
and 589 nm (I538/I589) was plotted as a function of the OCl- concentration (Fig. S2).
<Inserted Figure 5>
3.3. Effect of reaction time
We further examined the time courses of the fluorescence intensities of the probe in
the presence of 10 equiv. OCl- in the solution. As the Fig. 6 depicted, the ratio response
signal was reached within 15 s, indicating the fluorescence response of the probe was
fast. Therefore, reaction time of 60 s was selected for the subsequent experiments.
<Inserted Figure 6>
3.4. pH effects
In order to evaluate the possible applications of BDP-OX in different biological
environment, the ratiometric responses of the BDP-OX toward OCl- at different pH
conditions were investigated (Fig. 7). Within the pH range 5~10, obvious fluorescence
intensity ratio (I538/I589) variation could be observed.Considering the pKa of HOCl is 7.6,
BDP-OX can be used to detect both HOCl/OCl- successfully within a physiological pH
range.
<Inserted Figure 7>
3.5 Solvent effect for hypochlorite detection and the study UV-is and fluorescence
spectra of detecting in PBS buffer (pH 7.4, 20mM, 10% DMF).
Further, the basic spectroscopic properties of BDP-OX was studied in PBS buffer (pH
7.4, 20mM, DMF 10%). As the absorption spectra (Fig. 8a) depicted, the BDP-OX
exhibited amaximum absorption at 535 nm and the maximum absorption peak presented
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an obvious decreasing and a blue-shift with the addition of OCl- in PBS buffer (pH 7.4,
20mM, 10% DMF). The fluorescence spectra measurements was conducted by titrating
the aqueous solution of OCl-（2×10-3）gradually into the PBS buffer (pH 7.4, 20mM,
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10% DMF) containing 2.0 µM probe, at the same time, any changes in the fluorescence
intensity were monitored using a fluorescence spectrometer (λex = 488 nm, slit: 5 nm/5
nm). The fluorescent intensity of BDP-OX exhibited a series of remarkable changes，
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with the increasing concentrations of OCl- ions. The intensity of the emission maximum
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at 566 nm takes place blue shift and centered at 541 nm (Fig. 8b). Importantly, the
fluorescent intensity at 541 nm displayed a 20-fold increase. The emission intensities at
541 nm was plotted as a function of the OCl- concentration, there is a
proportional relation between fluorescent intensity and OCl- concentration (Fig. S3).
However, it is noted that ratiometric change did not appear. We also studied the
fluorescence spectra of detecting in CH3CN-PBS and ethanol-PBS buffer, the results are
similar to that in DMF-PBS (Fig. S6). These also proved that ratiometric fluorescence
depended on solvent.
<Inserted Figure 8 >
3.6 The Proposed Response Mechanism
The proposal mechanism for the detection of OCl- using the BDP-OX was interpreted
in Scheme 2. As the previous literature about oxidative-isomerization with hypochlorite
described, the oxime protective group could be transformed by OCl- to give the nitrile
oxide derivative [33]. Thus, “oxime” is transformed into “CN-O”. The emission of
compound “BDP-CNO” displayed a blue-shift relative to that of BDP-OX, due to
stronger ICT effect. Because the BODIPY is an electron-rich fluorophore and the
electron-withdrawing ability of the “CN-O” group is stronger than that of the oxime
group, the electrons are easy to transfer from BODIPY to “CN-O” group, accompanied
with the increasing of energy. These results are corresponding to the UV-Vis absorption
and fluorescent response and mass spectrometry.
<Inserted Scheme 2>
3.7. Cellular Imaging
Ability of BDP-OX for Detect Exogenous HOCl. To assess the capability of BDP-OX
to detect HOCl in cells, A549 cells stain with BDP-OX (2 µM) were treated with 10 µM
NaClO for 30 min. The fluorescence intensities from the green channel (λem = 525 ± 25
nm) clearly increased after NaClO stimulation (Fig. 9). As show in Fig. 9 (a1-a5)
brightfield and fluorescence images of the cells only incubated with the probe (2 µM)
for 30 min, from the red channel (a2) can observed strong red fluorescence, but green
channel (a3) there was nonfluorescence. Fig. 9 (b1-b5) brightfield and fluorescence
images of the cells incubated with the probe BDP-OX (2 µM) for 30 min, and then with
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10 µM NaOCl for 20 min, fluorescence image from the red channel (b2) can observed
relative weak red fluorescence to (a2), and from the green channel (b3) the green
fluorescence was appeared. Furthermore, the merged image (a4, b4) and ratio image of
the green to red channel (a5, b5) also demonstrated the changes, when BDP-OX treated
with NaOCl, the emission at 589 nm decreasing, and emission at 538nm increasing.
These cell experiments show the good cell-membrane permeability of probe, and it can
thus be used to imaging OCl- within living cells.
<Inserted Figure 9>
4. Conclusions
In summary, we fabricated a novel BODOPY-Dialdoxime (BDP-OX) based
fluorescent probe by integrating the BODIPY fluorophore with two aldoxime unit. This
probe exhibited a remarkable ratiometric fluorescent response to hypochlorite with the
emission the intensity displayed a 20-fold increase with this probe, with a fast response
fluorescent signal and a low micromolar detection limit. Additionally, this probe
exhibited higher selective for OCl- detection over other species due to distinct
isomerisation conditions, which the oxime was deprotected and transferd into the nitrile
oxide derivative. It’s a promising probe that can be used more effectively for imaging in
living cells. These successful/unique applications prospect that the BDP-OX will be a
powerful tool in the investigations of OCl- in living systems.
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Acknowledgments
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The work was supported by the National Natural Science Foundation of China (No.
21472118), the Program for the Top Young and Middle-aged Innovative Talents of
Higher Learning Institutions of Shanxi (2013802), Talents Support Program of Shanxi
Province (2014401), Shanxi province Outstanding Youth Fund (2014021002), another
is in the Shanxi University funds for study aboard 2014.
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Figure captions
Scheme 1 Synthesis of BDP-OX.
Fig. 1 Structure and thermal ellipsoids of probe are drawn at the 50% probability level
BDP-OX.
Fig. 2 The fluorescence intensity of BDP-OX (5 µM) with OCl- (10 µM) and other
various analytes (100 equiv.) in the PBS : DMSO = 1 : 9 (V/V pH = 7.4) (λex = 488 nm,
slit: 5 nm/5 nm).
Fig. 3 Competitive fluorescence signaling of OCl- to BDP-OX in the presence of
various metal ions as background. The BDP-OX (2 µM) with OCl- (10 µM) and other
mental ions (50 equiv.) in the PBS : DMSO = 1 : 9 (V/V pH = 7.4) (λex = 488 nm, slit: 5
nm/5 nm).
Fig. 4 UV-vis spectra of BDP-OX (10 µM) in PBS : DMSO = 1 : 9 (V/V pH = 7.4)
upon addition of various concentrations of OCl- (0-50 µM).
Fig. 5 Fluorescence spectra of BDP-OX (2 µM) in the presence of various
concentrations of OCl- in PBS : DMSO = 1 : 9 (V/V pH = 7.4) (λex = 488 nm, slit: 5
nm/5 nm). Inset: Visual fluorescence change photograph for BDP-OX only and upon
addition of NaClO (10 µM) under illumination with a 365 nm UV lamp.
Fig. 6 Reaction time profile of emission 546 nm BDP-OX (2 µM) and OCl- (20 µM) in
the PBS : DMSO = 1 : 9 (V/V pH = 7.4).
Fig. 7
pH effects.
Fig. 8 a) UV-vis spectra of BDP-OX (10 µM) in PBS buffer (pH 7.4, 20mM, 10% DMF)
upon addition of various concentrations of OCl- (0-10 µM); b) Fluorescence spectra of
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BDP-OX (2 µM) in the presence of variousconcentrations of OCl- in the PBS buffer (pH
7.4, 20mM, 10% DMF) (λex = 488 nm, slit: 5 nm/5 nm). Inset: Visual fluorescence
change photograph for BDP-OX only and upon addition of NaClO (10 µM) under
illumination with a 365 nm UV lamp.
Scheme 2 Proposed Response Mechanism and NMR Analysis
Fig. 9 (a1-a5) Incubated with the probe only: (a1) brightfield image; (a2) red channel;
(a3) green channel; (a4) merged images; (a5) ratio images; (b1-b5) Incubated with the
probe BDP-OX, and then treat with NaOCl: (b1) brighfield image; (b2) red channel; (b3)
green channel, (b4) merged images; (b5) ratio images. Excitation at 488 nm, the green
and red channels were set at 525 ± 25 nm and 595 ± 25 nm, respectively scale bar = 20
µm.
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Scheme 2
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Supporting Information
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Contents:
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1. Synthesis, and 1H NMR, 13C NMR, ESI-MS spectra and structure of the BDP-OX.
Fig.S1 ESI-MS analysis of BDP-OX in the present of NaClO.
Fig.S2 The ratios of emission intensities at the wavelength of 538 and 589 nm
(I538/I589) was plotted as a function of the OCl- concentration (0-50µM).
Fig.S3 The selectivity of BDP-OX toward OCl-.
Fig.S4 The Crystal data of the BDP-OX.
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Methods. All spectroscopic measurements were performed in DMSO-PBS (9:1, v:v, pH 7.4) solution. The
UV-Visible spectra and fluorescence spectra were recorded at 25 ℃. The decrease of the absorbance at 535 nm.
Measurement procedure The UV-Vis procedures were shown as follows: into a DMSO-PBS (9:1, v:v, pH 7.4)
containing 10µM BDP-OX, OCl- sample was gradually titrated. All UV-Vis spectra data were recorded at 1 min after
the OCl- addition.
The fluorescence procedures were as follows: into a DMSO-PBS (9:1, v:v, pH 7.4) solution containing 5.0 µM
BDP-OX, OCl- sample was gradually titrated. All fluorescence spectra data were recorded at 1 min after the OCl-
addition.
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1. Synthesis, and 1H NMR, 13C NMR, ESI-MS spectra and structure of the BDP-OX.
Synthesis of the BDP-OX.
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4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (1).
The benzaldehyde (0.424g, 4 mmol) and 2,4-dimethylpyrrole (0.833 g, 8.7 mmol) were dissolved in
dry CH2Cl2 (200 mL). Two drops of trifluoroacetic acid (TFA) were added to the reaction mixture,
and the resulting mixture was stirred in the dark for 12 h under nitrogen atmosphere at room
temperature.
After
the
complete
consumption
of
aldehyde,
DDQ
(2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (0.98 g, 4 mmol) was added to the reaction mixture.
When the mixture was stirred for 40 min, 9 mL of triethylamine and 9 mL of BF3·OEt2 were added
to the mixture. After the mixture was further stirred for 40 min, it was concentrated, and then was
washed twice with water, dried over anhydrous Na2SO4, and concentrated under reduced pressure.
The crude product was purified by column chromatography to obtain orange-red crystal solid 1 (0.38
g, 30%). 1H NMR (600 MHz, DMSO-d6): δ 7.57 (d, J =6.0 Hz, 3H), 7.38 (d, J = 6 Hz, 2H), 6.18 (s,
2H), 2.45 (s, 6H), 1.33 (s, 6H). 13C NMR (150 MHz, DMSO-d6): δ 155.31, 143.19, 142.41, 134.45,
131.14, 129.75, 128.18, 121.85, 14.69, 14.40. ESI-MS m/z: [M + H]+ Calcd for 324.16; Found
325.16.
4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-2-formyl-4-bora-3a,4a-diaza-sindacene (2).
A mixture of DMF (6 mL) and POCl3 (6 mL) was stirred in an ice bath for 5 min under nitrogen.
After the solution was warmed to room temperature, it was stirred for additional 30 min. To this
reaction mixture was added compound 1 (149 mg, 0.42 mmol) in dichloroethane (20 mL), the
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temperature was raised to 50 C, and the mixture was stirred for an additional 2 h. The reaction
mixture was cooled to room temperature and slowly poured into saturated aqueous NaHCO3 (300
mL) under ice-cold conditions. After being warmed to room temperature, the reaction mixture was
further stirred for 60 min and washed with water. The organic layers were combined, dried over
anhydrous Na2SO4, and evaporated in vacuo. The crude product was further purified using column
chromatography (silica gel, hexane/EtOAc/CH2Cl2, 4/1/1, v/v) to give BODIPY 2 (142 mg, 89%) as
red crystal solid.1H NMR (600 MHz, DMSO-d6): δ 9.95 (s, 1H), 7.60 (s, 3H), 7.43 (s, 2H), 6.45 (s,
1H), 2.71 (s, 3H), 2.55 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ 187.09,
162.16, 155.56, 147.61, 144.10, 142.26, 133.79, 130.16, 129.97, 129.43, 128.13, 126.38, 124.91,
15.21, 13.11, 11.59. ESI-MS m/z: [M + H]+ Calcd for 352.19; Found 352.92.[M+Na]+ Calcd for
374.18; Found 374.83.
4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-2,6-diformyl-4-bora-3a,4a-diaza-sindacene (3).
A mixture of DMF (6 mL) and POCl3 (6 mL) was stirred in an ice bath for 5 min under nitrogen.
After the solution was warmed to room temperature, it was stirred for additional 30 min. To this
reaction mixture was added compound 2 (160 mg, 0.42 mmol) in dichloroethane (20 mL), the
o
temperature was raised to 60 C, and the mixture was stirred for an additional 5 h. The reaction
mixture was cooled to room temperature and slowly poured into saturated aqueous NaHCO3 (300
mL) under ice-cold conditions. After being warmed to room temperature, the reaction mixture was
further stirred for 60 min and washed with water. The organic layers were combined, dried over
anhydrous Na2SO4, and evaporated in vacuo. The crude product was further purified using column
chromatography (silica gel, hexane/EtOAc/CH2Cl2, 4/1/1, v/v) to give BODIPY 3 (141 mg, 82%) as
red crystal solid. 1H NMR (600 MHz, CDCl3): δ 10.06(s, 2H), 7.60 (s, 3H), 7.31 (s, 2H), 2.89 (s, 6H),
1.71 (s, 6H). 13C NMR (150 MHz, CDCl3): δ 185.55, 160.75, 148.39, 147.26, 133.58, 133.55,
131.87, 130.18, 130.16, 129.91, 129.88, 128.10, 127.39, 127.36, 13.80, 12.03. ESI-MS m/z: [M +
H]+ Calcd for 353.19; Found 352.92. [M + Na]+ Calcd for 375.18; Found 374.83.
Synthesis of 4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-2,6-dioxime-4-bora-3a,4a –diaza-sindacene
(BDP-OX).
Compound 3 (114 mg, 0.3 mmol) and hydroxylamine hydrochloride (41.4 mg, 0.6 mmol) in absolute
ethanol (10.0 mL) were stirred at reflux temperature for one hour. After the completion of reaction
the solvent was removed under reduced pressure and the residue was purified by column
chromatography (hexane/ethyl acetate (4/1)). BODIPY-dioxime was obtained as red solid (86.0 mg,
1
70.0% yield). H NMR (600 MHz, CDCl3) δ: 11.17(s, 2H), 8.08(s, 2H), 7.60(s, 3H), 7.42(s, 2H),
2.64(s, 6H), 1.43(s, 6H). 13C NMR (150 MHz, CDCl3): δ 167.42, 155.65, 143.51, 142.85, 134.26,
132.16, 131.99, 131.07, 130.07, 129.93, 129.13, 128.34, 123.98, 30.47, 19.12, 14.48, 12.65. ESI-MS
m/z: [M + H ]+ Calcd for 381.20; Found 380.92. Crystal size: 0.200 x 0.160 x 0.090 mm,
monoclinic, space group P 2(1)/c. a=9.1318 (6) Å, b= 13.7392(9) Å, c=16.5704(11) Å, α= 90o,
β=103.270(2)o. γ= 90o. V =2023.5(2) Å3，Z= 4, T=300(2) K, θmax=28.298o. 19891 refections
measured, 5007 unique (Rint = 0.0511). Final residual for 277 parameters and 5007 refections with I
> 2σ (I): R1 = 0.0599, wR2 = 0.01361 and GOF = 1.018.
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The H NMR (600MHz) spectra of the 4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-4-bora -3a,4a-diaza-s-indacene
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The 13C NMR (150 MHz) spectra of the 4,4-Difluoro-8-(phenyl)-1,3,5,7-tetramethyl-4-bora
-3a,4a-diaza-s-indacene in DMSO-d6.
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