A benzothiazole-based fluorescent probe for hypochlorous acid detection and imaging in living cells

Article abstract of DOI:10.1016/j.saa.2018.03.055

A benzothiazole-based turn-on fluorescent probe with a large Stokes shift (190 nm) has been developed for hypochlorous acid detection. The probe displays prompt fluorescence response for HClO with excellent selectivity over other reactive oxygen species as well as a low detection limit of 0.08 μM. The sensing mechanism involves the HClO-induced specific oxidation of oxime moiety of the probe to nitrile oxide, which was confirmed by HPLC-MS technique. Furthermore, imaging studies demonstrated that the probe is cell permeable and can be applied to detect HClO in living cells.

Full text of DOI:10.1016/j.saa.2018.03.055

Accepted Manuscript
A benzothiazole-based fluorescent probe for hypochlorous acid
detection and imaging in living cells
Khac Hong Nguyen, Yuanqiang Hao, Ke Zeng, Shengnan Fan,
Fen Li, Suke Yuan, Xuejing Ding, Maotian Xu, You-Nian Liu
PII:
S1386-1425(18)30263-4
doi:10.1016/j.saa.2018.03.055
SAA 15927
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 9 February 2018
Revised date:
Accepted
date:
17 March 2018
2
3 March 2018
Please cite this article as: Khac Hong Nguyen, Yuanqiang Hao, Ke Zeng, Shengnan Fan,
Fen Li, Suke Yuan, Xuejing Ding, Maotian Xu, You-Nian Liu , A benzothiazole-based
fluorescent probe for hypochlorous acid detection and imaging in living cells. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Saa(2017), doi:10.1016/j.saa.2018.03.055
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ACCEPTED MANUSCRIPT
A benzothiazole-based fluorescent probe for hypochlorous acid detection and
imaging in living cells
a, b
b,
a
b
b
Khac Hong Nguyen , Yuanqiang Hao , Ke Zeng , Shengnan Fan , Fen Li , Suke
b
b
b
a,
Yuan , Xuejing Ding , Maotian Xu , and You-Nian Liu
a
College of Chemistry and Chemical Engineering, Central South University,
Changsha, Hunan 410083, P. R. China
b
Henan Key Laboratory of Biomolecular Recognition and Sensing, College of
Chemistry and Chemical Engineering, Shangqiu Normal University, Shangqiu,
Hunan 476000, P. R. China

Corresponding authors. Fax/Phone: +86-370-2586802.
E-mail addresses: hao0736@163.com (Y. Hao), liuyounian@csu.edu.cn (Y.-N. Liu).
1

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Abstract
A benzothiazole-based turn-on fluorescent probe with a large Stokes shift (190 nm)
has been developed for hypochlorous acid detection. The probe displays prompt
fluorescence response for HClO with excellent selectivity over other reactive oxygen
species as well as a low detection limit of 0.08 μM. The sensing mechanism involves
the HClO-induced specific oxidation of oxime moiety of the probe to nitrile oxide,
which was confirmed by HPLC-MS technique. Furthermore, imaging studies
demonstrated that the probe is cell permeable and can be applied to detect HClO in
living cells.
Keywords: Fluorescent probe, Large Stokes shift, Hypochlorous acid, Cell imaging,
Benzothiazole
2

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1
. Introduction
Reactive oxygen species (ROS) have received an increasing attention due to their
essential roles in biology and their relevance to many diseases [1, 2]. Among the
known ROS, hypochlorous acid (HClO), which is biologically produced from
–
hydrogen peroxide (H O ) and chloride ion (Cl ) enzymatically by MPO
2
2
(myeloperoxidase), acts as a powerful microbicide agent in the innate immune
systems during pathogen invasion [3]. In addition, as a highly potent oxidant, HClO
can also react with amino acids, proteins, cholesterol and nucleosides, resulting in the
inhibition of various biomolecular functions [4, 5]. Moreover, it is reported that
abnormal production of HClO is closely related to a number of diseases, including
cardiovascular disease [6], inflammatory disease [7], certain cancers [8] and
neurodegenerative disorders [9]. Therefore, the development of accurate and reliable
techniques for HClO detection and imaging in living cells is of great important to
better understanding its physiological and pathological functions.
Fluorescence imaging based on synthetic probes is a powerful tool for sensing
HClO both in vivo and in vitro due to its unique advanteges, such as high sensitivity,
capability for spatial and temporal resolution, simplicity for implementation, and rea-
time analysis [10-14]. As a potent oxidant and chlorination agent, HClO can mediate
specific reactions towards a number of moieties [15, 16], such as chalcogenide (S, Se,
Te) atoms, dibenzoylhydrazine, acylhydrazone, hydroxylamine, hydrazone, Schiff
bases, electron deficient alkenes, p-methoxyphenol, 1,8-diaminonaphthalene and so
on. On the basis of these specific reactivities of HClO, a variety of fluorescent probes
have been reported for the detection of HClO by using fluorophores, including
oxazine [17], Si-rhodamine [18], fluorescein [19-22], BODIPY [11, 23, 24], acedan
[
10], cyanine dye [25], fluorenone [26], coumarin [27], iridium(III) complex [28],
3

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naphthalimide [29-32], phenanthroimidazole [33], (2-hydroxyphenyl) benzothiazole
(HBT) [34], 7-nitro-2,1,3-benzoxadiazole [35]. Among these utilized fluorohpores,
HBT and its derivatives have gained a great deal of interets due to their abilities to
undergo an excited-state intramolecular photon transfer (ESIPT) process upon
photoexcitation through the keto-enol tautomerism which results in a large Stokes
shift and/or unique ratiometric fluorescence behaviour [36-38].
Inspired by these insights, we reported a HBT-based turn on fluorescent probe for
HClO detection. The probe was obtained by condensation of HBT with NH –OH.
2
Due to the non-radiative deactivation via C=N isomerization, the probe is weakly
fluorescent. The specific HClO-mediated formation of the nitrile oxide could generate
a highly emissive derivative. This transformation of the probe induced by HClO was
confirmed by high performance liquid chromatography-mass spectrometry (HPLC-
MS) technique. The new probe exhibits high sensitivity with a low detection limit of
0
.08 μM, as well as excellent selectivity for HClO over other relevant species.
Moreover, the probe displays excellent cell membrane permeability and low cell
cytotoxicity, and was successfully applied to image HClO in living cells.
2
. Experimental
2
.1. Materials and instrumentations
All reagents were purchased from commercial suppliers and used as received.
Deionized water was used throughout the analytical experiments. The fluorescence
spectra were carried out on a Varian Cary Eclipse fluorescence spectrophotometer
(Agilent Technologies, Santa Clara, USA). The absorbance spectra were recorded
with a Cary 60 UV-Vis spectrophotometer Agilent (Agilent Technologies, Santa
4

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Clara, USA). NMR spectra were recorded on a Bruker 400 MHz. Mass spectrometry
was performed on a Xevo G2-S Q-TOF mass spectrometer (Waters, Milford, MA,
USA). HPLC analysis was performed on a Waters Acquity UPLC H-Class system
(Milford, MA, USA) equipped with a quaternary solvent delivery system, a column
oven, an auto sampler and a photodiode array detector. The analytes were separated in
gradient mode with a Waters Acquity BEH 2.1 × 50 mm C18 1.7 μm column. The
o
column oven was kept at 40 C. Flow rate was 0.3 mL/min. Eluent components were
water (A) and acetonitrile (B). The mobile phase gradient was as follows: 0.0 min 50%
B → 4.0 min 100 % B→ 5.0 min 50 % B.
2
.2. General procedure for analysis
A parent stock solution of probe 1 (10 mM) was prepared in absolute ethanol. Test
solution of probe 1 was diluted in a mixed solution of phosphate buffer: C H OH =
2
5
9
:1 (v/v, pH 7.4, 10 mM) and small aliquots of each testing species solution were
added. All spectra were obtained in a quartz cuvette (path length = 1 cm) at room
temperature.
2
.3. Fluorescence Imaging
The cultured Hela cells were incubated with probe 1 (10 μM), probe 1 (10 μM) and
–
consequently with ClO (20 μM) in DMEM (Dulbecco's modified Eagle medium) at
o
3
7 C, respectively. The incubation time was set at 1 h. After incubation for the
corresponding time, the cells were washed with PBS three times to remove free
compound and ions before analysis. Fluorescence imaging was performed with an
Olympus IX 71 with xenon lamp and Olympus digital camera. Fluorescence in green
channels was visualized with the appropriate emission filters.
2
.4. Cell viability assay
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The toxicity of probe 1 towards living cells was determined by MTT (3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assays. Three kinds of
cell liens (HeLa, L929, and MDA-MB-231 cells) were evaluated. The cells were
3
seeded in 96-well plates at a density of 5 × 10 cells per well and cultured for 24 h at
o
3
7 C with 5% CO in humidified environment. Then a series of different
2
concentrations of probes (0, 5, 10, 15, and 20 μM) were added to the separated wells,
and the cells were incubated for an additional 24 h. MTT solution was then added into
each well and then residual MTT solution was removed after 4 h. The MTT-formazan
crystals were dissolved in 200 μL DMSO. The absorbance of each well was measured
by a microplate reader (Bio-TekELx800) at the wavelength of 490 nm. The cell
viability was assessed using the following Equation:
Cell viability (%) = A /A × 100%
T
0
where A is the absorbance of treated cells and A is the control absorbance. Data of
T
0
cell viability are given as mean ± standard deviation (S.D.) (three replicate
measurements).
2
.5. Synthesis of probe 1
The preparation of probe 1 was shown in scheme 1. Compounds 2 and 3 were
synthesized according the previously reported procedures [39].
Compound 2 (0.135 g, 0.5 mmol) and hydroxylamine hydrochloride (0.069 g, 1.0
mmol) was dissolved in absolute ethanol (30 mL). Triethylamine (0.11 g, 1.1 mmol)
o
was added to the solution, and then the mixture was refluxed under 80 C for 5 h. The
volatiles were then removed under reduced pressure, and the residue was taken up in
DCM (dichloromethane) and washed with H O for three times. The organic layer was
2
6

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dried over Na SO , filtered, and then dried under vacuum. Purification via column
2
4
chromatography using DCM/MeOH as eluent (100:3, v/v) afforded 1 as a white solid
1
(
(
(
0.183 mg, 65%). H NMR (400 MHz, DMSO): δ 12.20 (s, 1H), 11.63 (s, 1H), 8.46
s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 7.98 (s, 1H), 7.55 (s, 2H), 7.49 (s, 1H), 2.36 (s, 3H)
1
3
see Fig. S1). C NMR (101 MHz, DMSO) δ 165.68, 153.28, 151.68, 147.40, 134.38,
1
31.73, 130.04, 129.29, 127.18, 125.93, 122.66, 122.60, 120.19, 118.83, 20.39 (see
+
Fig. S2). MS [ESI]: m/z, calcd for [M+H] 258.06; found 285.01 (see Fig. S3).
Scheme 1 Synthesis of probe 1.
3
. Results and Discussion
–
3
.1. Spectral response of probe 1 to ClO
–
The spectral responses of probe 1 towards ClO were investigated by both UV-Vis
and fluorescence titration in a H O-EtOH solution (9:1, v/v, 10 mM phosphate buffer,
2
pH 7.4). As shown in Fig. 1, Probe 1 displays a main absorption band around 350 nm.
–
Upon addition of ClO , a weak shoulder absorption band at 410 nm was gradually
emerged with a slight decrease of originally absorption at 350 nm. This bathochromic
shift can be attributed to the enhanced ICT (intramolecular charge transfer) process
–
within the oxidized product of 1 by ClO , as the generated nitrile oxide is a stronger
electron-withdrawing group compared with the unreacted hydroxylamine. Fig. 1B
–
shows the fluorescent responses of probe 1 to ClO . Probe 1 exhibits weak
fluorescence in the absence of HClO at the excitation of 350 nm due to the C=N
isomerization involved decay process. The introduction of HClO into the solution of 1
7

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led to an obvious fluorescence enhancement at 540 nm, which can be ascribed to the
–
conversion of the quenching group of hydroxylamine by ClO . Additionally, a good
linear relationship was obtained between the fluorescence intensities at 540 nm and
the HClO concentrations in a range of 0.5–18.0 μM (inset in Fig. 1B). The detection
limit was estimated to be 0.08 μM based on 3σ. The performance of probe 1 is
–
compared with various hydroxylamine-based fluorescent ClO (listed in Table S1).
The proposed probe exhibits comparable or superior analytical performance in the
aspects of detection medium, Stokes shift and detection limit.
Fig. 1. UV-Vis spectra (A) and fluorescence spectra (B) of probe 1 (10 µM) in H O-
2
EtOH solution (9:1, v/v, 10 mM phosphate buffer, pH 7.4) upon addition of various
–
concentrations of ClO (0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0 18.0, and
2
0.0 μM). Inset shows the linear fitting curve between the fluorescence intensity at
–
5
40 nm and the concentration of ClO . λ = 350 nm.
ex
Subsequently, the time-dependence of fluorescence response of the probe for HClO
–
was evaluated. In the presence of ClO , the probe solution reveals a dramatic
enhancement in the emission intensity at 540 nm (Fig. S4). Under pseudo-first-order
–
3
–1
conditions, the reaction rate constant (k’) was calculated to be 3.9 × 10 s . And the
8

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emission intensity reached the maximum in 15 min, indicating that probe 1 has the
–
capability for real-time detection of ClO . The selectivity of probe 1 toward various
ROS and other interferences was investigated. The probe possesses a 100-fold higher
–
t
3+
1
response for ClO over other species, such as BuOOH, Fe , H O , NO, O , •OH,
2
2
2
–
•
O and TBHP (tert-butyl hydroperoxide), indicating that probe 1 is highly selective
2
–
–
for ClO (see Fig. 2). This specific fluorescence response of probe 1 towards ClO also
can be visualized under a Handheld UV Lamp (Fig. 2B, inset).
Fig. 2. (A) Fluorescence spectra of probe 1 (10 µM) in H O-EtOH solution (9:1, v/v,
2
1
0 mM phosphate buffer, pH 7.4) in the presence of various ROS and other
interferences (20 µM); (B) Fluorescence intensity changes at 540 nm (λ = 350 nm).
ex
The inset shows the corresponding visual fluorescence change photograph of the
–
probe solution for ClO and interferences under a Handheld UV Lamp (λ = 365 nm).
ex
3
.2. Sensing mechanism
9

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–
Fig. 3. Proposed sensing mechanism of probe 1 towards ClO and the frontier orbital
diagram of probe 1 and its speculated products.
Hydroxylamine has been successfully employed as a recognition moiety for the
–
construction of fluorescent ClO probes [16]. But this hydroxylamine group in
–
different probes can probably be oxidized into different species by ClO , such as
aldehyde [40-42], nitrile oxide [43, 44], carboxylic acid [45, 46]. To better understand
–
the sensing mechanism of probe 1 towards ClO , the reaction process was monitored
by HPLC-MS (see Fig. S5). The HPLC chromatogram of probe 1 shows a single peak
with retention time (Rt) at 3.17 min, which corresponds to a m/z value of 258.01.
–
After incubation with ClO (1 equiv.) for 15 min (Fig. S5B), a new chromatographic
peak was emerged (R = 3.60 min) with a m/z value of 252.99, which can be ascribed
t
to the generated nitrile oxide derivative (cal. 253.05). These HPLC-MS results clearly
indicates the conversion of probe 1 into its nitrile oxide product as depicted in Fig. 3.
To further inspect the relationship between the electronic structure changes and
–
optical response of probe 1 to ClO , a theoretical calculation was carried out by
Density Functional Theory (DFT) with the B3LYP/6-31+G(d,p) method basis set
using the Gaussian 09 program. Fig. 3 presents the frontier orbital diagram, highest
occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital
1
0

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(
LUMO), of probe 1 and the corresponding products. Due to the generated nitrile
oxide group, the product displays decreases in the energy levels of both LOMO and
HOMO, and a smaller energy gap, in good agreement with the red shift of the UV-Vis
spectra.
3
.3. Cellular imaging
Fig. 4. Fluorescence images of live Hela cells. Cells incubated with 10 µM probe 1
–
for 60 min at 37 °C (A, B). Probe 1 loaded cells after treatment with 20 µM ClO for
6
0 min at 37 °C (C, D). (A) and (C) are bright-field images. Scale bar represents 20
μm in all images.
–
We further sought to apply probe 1 for fluorescence imaging of ClO in living cells.
o
HeLa cells loaded with 10 µM probe 1 for 1 h at 37 C showed faint fluorescence
–
indicate that the probe is cell-permeable (Fig. 4B). Addition of 20 µM ClO to HeLa
1
1

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cells loaded with probe 1 led to a significant increase in intracellular fluorescence
–
compared to control cells without treatment by ClO (Fig. 4D). Furthermore, the
cytotoxicities of probe 1 towards different types of cells were evaluated using MTT
assay. All these inspected cells remained in good condition after 24 h incubation with
various concentrations (0–20 μM) of probe 1 (Fig. S6), implying the low cytotoxicity
of probe 1. These cell experiments demonstrate that probe 1 is biocompatible, cell
–
membrane permeable, and can be utilized for monitoring ClO in live cells.
4
. Conclusion
–
In summary, a new turn-on fluorescent probe (1) for ClO by condensation of
aldehyded HBT fluorophore with the recognition moiety of hydroxylamine was
–
presented. Probe 1 displays sensitive and selective fluorescence response toward ClO
–
over other ROS via the ClO -induced specific transformation of the quenching group
of hydroxylamine into nitrile oxide within probe 1. The reaction mechanism was
confirmed by HPLC-MS. Furthermore, probe 1 exhibits excellent biocompatibility
–
and cell membrane permeability, and can be utilized for monitoring ClO in live cells.
This novel probe provides a new model for detection and bioimaging of ROS in living
cells.
1
2

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ACKNOWLEDGMENTS
We are grateful to the National Natural Science Foundation of China (Nos. B061201,
U1404215, 21475084, 21476266, and 21475085) and Innovation Scientists and
Technicians Troop Construction Projects of Henan Province (No: 41) for support.
1
3

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Graphical Abstract
1
8

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Highlights
–




A new benzothiazole-based turn-on fluorescent probe 1 was developed for ClO .
–
Probe 1 features high sensitivity (LOD: 0.08 μM) and selectivity to ClO .
The sensing mechanism was confirmed by HPLC-MS technique.
–
Probe 1 was utilized for monitoring ClO in live cells.
1
9