Benzothiazoline based chemodosimeters for fluorogenic detection of hypochlorous acid

Article abstract of DOI:10.1016/j.bmcl.2013.05.083

Two nonfluorescent and colorless chemodosimeters featuring benzothiazoline moiety were developed for chromo-fluorogenic detection of HOCl.

Full text of DOI:10.1016/j.bmcl.2013.05.083

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4354–4357
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzothiazoline based chemodosimeters for fluorogenic detection
of hypochlorous acid
Zhisheng Wu a, , Xuanjun Wu a, , Zhu Li a, Yuhui Yang a, Jiahuai Han b, Shoufa Han a,
⇑
a Department of Chemical Biology, College of Chemistry and Chemical Engineering, and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University,
Xiamen 361005, China
b State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen 361005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two nonfluorescent and colorless chemodosimeters featuring benzothiazoline moiety were developed
for chromo-fluorogenic detection of HOCl.
Received 26 April 2013
Revised 23 May 2013
Accepted 24 May 2013
Available online 7 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Rhodamine
Benzothiazoline
Hypochlorous acid
Bioimaging
Fluorogenic
Hypochlorous acid (HOCl), with pKa of 7.5, is poised to dissoci-
ate into hypochlorite (OClÀ) in aqueous media. Artificial HOCl or
hyperchlorite is widely used as disinfection agents in civilized
society. Biogenic HOCl, synthesized from hydrogen peroxide and
chloride via the catalysis of myeloperoxidase in leukocytes, could
also be directed against infectious microorganisms by the host
immune systems. Despite its protective roles, abnormal levels of
HOCl in vivo have been associated with multiple diseases including
arthritis, neuron degeneration and cancers.1 Imaging agents that
allow facile detection or fluorescent tracking of HOCl will be
valuable for investigations on HOCl related biological processes
or monitoring the levels of HOCl in abiotic samples, for example,
drinking water. The multiple roles of HOCl in the aforementioned
phatho-physiological events had inspired the development of a
number of chemodosimeters for HOCl detection via analyte trig-
gered oxidation of integrated HOCl-responsive fuctionalities
including hydroxamate, hydrazide, and thioester, etc.2 Here we
report the fluorogenic detection of HOCl with two benzothiazo-
line-based chemodosimeters. Specifically, SA-thiazoline, conjugate
of salicylaldehyde with 2-aminothiophenol, undergoes HOCl
mediated oxidation of the thiazoline to give SA-thiazole which is
a conventional fluorophore employed in a number of studies.3 In
contrast, the rhodamine B-benzothiazoline diad (referred to as
RB-thiazoline) gives highly fluorescent and deep colored species
via oxidative opening of the intramolecular C–N bond (Scheme 1).
RB-thiazoline and SA-thiazoline, colorless and nonfluorescent,
were respectively prepared via condensation of 2-aminothiophe-
nol with 2-hydroxybenzaldehyde or rhodamine-aldehyde (Supple-
mentary data). We first examined the influence of solvents on the
fluorogenic reactions and identified that RB-thiazoline preferred
polar organic solvents whereas SA-thiazoline was applicable in
A
N
O
N
N
O
N
HOCl
N
N
S
S
RB-thiazoline
Nonfluorescent
RB-thiazole
Fluorescent and deep colored
OH
N
B
OH
H
N
HOCl
S
S
SA-thiazole
Fluorescent
SA-thiazoline
⇑
Corresponding author. Tel.: +86 592 2181728.
E-mail address: shoufa@xmu.edu.cn (S. Han).
Nonfluorescent
These authors contributed equally to this work.
Scheme 1. Detection of HOCl with RB-thiazoline (A) and SA-thiazoline (B).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.083

Z. Wu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4354–4357
4355
tions were recorded by fluorometry. Figure 2A showed that fluo-
rescence emission of RB-thiazole centred at 590 nm intensified as
a function of analyte concentrations. The highly fluorescent RB-thi-
azole is of deep red color, which indicated its utility in visual esti-
mation of HOCl in selected samples, for example, tap water. The
fluorescence emission of SA-thiazole, maximal at 462 nm, propor-
tionally increased as a function of HOCl concentration (Fig. 2C and
500
400
300
200
100
0
A
B
60 -M
10 -M
3000
2000
1000
0
60 -M
10 -M
0 -M
0 -M
0
100 200 300 400 500 600
Time (seconds)
0
100 200 300 400 500 600
Time (seconds)
D). In both assays, 10 lM of HOCl can be detected.
Reactive oxygen species (ROS) could be generated by mamma-
lian cells in responses to environmental stress. Demonstrated to be
responsive to HOCl, RB-thiazoline and SA-thiazoline were further
evaluated for their selectivity for other representative ROS. Figure
3 showed that SA-thiazoline and RB-thiazoline were both inert to
Figure 1. Kinetic profiles of the reactions between NaOCl and RB-thiazoline or SA-
thiazoline. (A) The fluorescence emission of RB-thiazole (10 M) in ethanol
containing NaOCl (0, 10, or 60 M) was recorded as function of time
(kem@590 nm; kex@560 nm); (B) time correlated fluorescence emission of SA-
thiazole (20 M) at 462 nm in phosphate buffered saline (PBS)/ethanol (50%, v/v)
containing NaOCl (0, 10 or 60 M) was recorded using kex@402 nm.
l
l
a
l
H2O2, nitric oxide (NO), ÅOH, ROOÅ, and ÅOÀ. 2-(2-Pyridyl)benzo-
l
2
thiazoline was used for fluorogenic detection of superoxide anion.4
It was shown that SA-thiazoline was highly responsive to HOCl as
compared to superoxide anion radical. (Fig. 3 and Fig. S4, Supple-
mentary data). Historically, rhodmaine-deoxylactams, derivatives
of rhodamine featuring intramolecular deoxylactams have been
used to stain lysosomes with acidic intracompartmental pH in live
cells by proton mediated opening of the intramolecular deoxylac-
tams.5 We therefore probed the fluorogenic responses of SA-thiaz-
oline and RB-thiazoline to pH and various ions that are often
present in biological specimens. It was shown that SA-thiazoline
exhibited negligible fluorescence towards all ions tested which in-
cluded H+, Na+, K+, Ca2+, Mg2+, Fe2+, Fe3+, Mn2+, Zn2+, Co2+, Cl À,
HPO24À, and HCO3À and SO24À (Fig. 3B), indicating the stringent selec-
tivity of SA-thiazoline for HOCl. In contrast, RB-thiazoline dis-
played significant fluorogenic response to Fe3+ ( Fig. 3A) and
proton (pH 4), presumably due to cation promoted opening of
the intramolecular deoxylactam. Taken together, the findings
showed that RB-thiazoline with moderate preference for HOCl
whereas SA-thiazoline is highly selective for HOCl, indicating its
utility for HOCl imaging in biological specimens.
aqueous media. Time course monitoring of formation of fluores-
cent species in the optimized assay solutions by fluorometry
showed that NaOCl triggered oxidation of the chemodosimeters
was completed immediately upon addition of various levels of
NaOCl (Fig. 1).
To ascertain the identities of the fluorescent species generated
under the optimized assay conditions, the assay solutions of RB-
thiazoline and SA-thiazoline were respectively, analyzed by mass
spectrometry (MS). A major signal at 532.2412 was identified in
the assay solution of RB-thiazoline (Fig. S1, Supplementary data),
confirming the genesis of RB-thiazole (C34H34N3OS+; MW:
532.2417). In a parallel experiment, generation of SA-thiazole
was also confirmed by MS (Fig. S2, Supplementary data). SA-thia-
zole was isolated by silica gel chromatography and then analyzed
by 1H NMR and 13C NMR (Supplementary data). Collectively, these
results supported the proposed HOCl-mediated oxidation mecha-
nisms as described in Scheme 1.
To probe the assay sensitivity, RB-thiazoline and SA-thiazoline
were respectively, added into a serial of solutions containing varied
levels of NaOCl. The fluorescence emission intensities of the solu-
To probe the impact of the hydroxyl moiety of SA-thiazoline in
sensing of HOCl, benzyl-thiazoline, which is an structural analog of
Figure 2. Titration of HOCl with RB-thiazoline and SA-thiazoline. (A) The fluorescence emission spectra of RB-thiazoline (10
(60, 50, 40, 30, 20, 10, and 0 M, from top to bottom) (kex@560 nm); the insert showed the visual images of the assay solution of RB-thiazoline before and after addition of
NaOCl (60 M); (B) titration curve of RB-thiazoline based assay was plotted by fluorescence emission intensity@590 nm versus NaOCl concentrations; (C) fluorescence
emission of SA-thiazoline (20 M) in PBS buffered ethanol (1:1, v/v) spiked with NaOCl (100, 80, 60, 40, 20, 10, and 0 M; from top to bottom) (kex@402 nm); the insert
M) under UV-illumination; (D) the titration curve of SA-thiazoline was plotted using
lM) in ethanol containing various levels of NaOCl
l
l
l
l
showed images of SA-thiazole solution with or without addition of NaOCl (60
l
fluorescence emission intensity@462 nm as a function of NaOCl concentrations.

4356
Z. Wu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4354–4357
A
6000
5000
4000
3000
2000
1000
N
O
N
N
S
0
700
600
500
400
300
200
100
0
B
HO
HN
S
Figure 5. Imaging of HOCl with SA-thiazoline in L929 cells. L929 cells were first
cultured with SA-thiazoline (0.5 mM) and then separately incubated in PBS
supplemented with or without NaOCl (10 M). The cells were washed with PBS
and then visualized by confocal fluorescence microscopy. The fluorescence emis-
sion@425–485 nm was collected with kex@405 nm.
l
We examined the efficacy of both probes for imaging HOCl in
living cells. L929 cells pre-cultured with SA-thiazoline were
respectively, incubated in PBS supplemented with or without
NaOCl. The cells were rinsed with PBS and then analyzed by confo-
cal fluorescence microscopy. The fluorescence of SA-thiazole was
clearly observed in cells treated with NaOCl relative to control cells
(Fig. 5), demonstrating the penetration of exogenous NaOCl into
cells which is consistent with previous studies2 and the utility of
SA-thiazoline in sensing of HOCl in live cells. Surprisingly, cells cul-
tured with RB-thiazoline exhibited strong rhodamine fluorescence
in the presence or absence of exogenous NaOCl. To probe the
analyte independent intracellular fluorescence of RB-thiazole,
L929 cells were co-stained with rhodamine 123 and RB-thiazoline.
Visualization of cells with confocal fluorescence microscopy
showed that the fluorescence of RB-thiazoline inside cells colocal-
ized with that of rhodamine 123 which is an established fluores-
cent marker for mitochondria (Fig. S5, Supplementary data).
Mitochondria are ubiquitous subcellular organelles hallmarked
by negative electrical potential across the mitochondrial mem-
brane which leads to intramitocondrial accumulation of lipophilic
and cationic species, for example, rhodamine 123.7 Staining of
mitochondria with RB-thiaozline suggested that RB-thiazoline
was turned into cationic fluorescent species, presumably due to
metal ion promoted opening of the intramolecular C–N bond to
give cationic species structurally resembling rhodamine 123.
In summary, fluorogenic detection of HOCl was achieved via
analyte mediated oxidation of two benzothiazoline-based chemo-
sodimeters. RB-thiazoline displayed sharp color changes upon
HOCl triggered oxidative opening of the intramolecular deoxylac-
tam and was suitable for visual detection of HOCl in Fe3+ free
neutral media. SA-thiazoline exhibited stringent selectivity for
HOCl and was shown to effectively sense HOCl in living cells. The
successful detection of HOCl with SA-thiazoline and RB-thiazoline
revealed the utility of benzothiazoline as efficient activatable
handle that can be integrated with appropriate signal generating
platforms in design of chemodosimeters targeting HOCl.
Figure 3. Selectivity of RB-thiazoline and SA-thiazoline for HOCl. To the solution of
RB-thiazoline (10 M) in ethanol (A) and SA-thiazoline (20 M) in PBS buffered
ethanol (1:1); (B) were respectively, added each of the following species (1 mM):
l
l
H2O2, ÅOH, ROOÅ, NO, O2ÀÅ, K+, Na+, Co2+, Mn2+, Mg2+, Ca2+, Zn2+, Fe3+, Fe2+, ClÀ, HPO42À
,
HCOÀ3 , SO24À, or NaOCl (0.1 mM). Fluorescence emission intensities@462 nm of SA-
thiazole were recorded using kex@402 nm whereas the fluorescence emission
intensities of RB-thiazole (kem@590 nm) were recorded using kex@560 nm.
SA-thiazoline, was synthesized following published procedure6
and tested under identical conditions. It was shown that no
fluorescent species was produced in the assay solution of benzyl-
thiazoline spiked with NaOCl (Fig. 4). The differential responses
of benzyl-thiazoline to HOCl as compared to that of SA-thiazoline
highlight the essential role of the hydroxyl group of SA-thiazoline
in fluorogenic sensing of NaOCl.
800
700
600
500
400
300
200
100
HO
HN
HN
S
S
0
- NaOCl + NaOCl - NaOCl + NaOCl
Acknowledgments
Figure 4. Fluorescence responses of benzyl-thiazoline to HOCl as compared to SA-
thiazoline. The fluorescence emission of benzyl-thiazoline (20 M) or SA-thiazoline
(20 M) in PBS buffered ethanol (1:1, v/v) spiked with or without NaOCl (100 M)
was recorded using kex@402 nm.
l
Dr. S. Han was supported by grants from NSF China 21272196,
21072162, Natural Science Foundation of Fujian Province
l
l

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Supplementary data
Supplementary data associated (synthesis and characterization
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