A strategy for highly selective detection and imaging of hypochlorite using selenoxide elimination

Article abstract of DOI:10.1021/ol4006823

A new strategy for HOCl-specific fluorescent probes has been reported based on a selenoxide elimination reaction. Probes CM1 and CM2 were synthesized as the first fluorescent probes containing an arylseleno moiety for hypochlorite according to this strategy. Both probes displayed excellent properties, including high selectivity and sensitivity, fast response, and pH independency toward hypochlorite in vitro and vivo.

Full text of DOI:10.1021/ol4006823

ORGANIC
LETTERS
2013
Vol. 15, No. 8
2002–2005
A Strategy for Highly Selective Detection
and Imaging of Hypochlorite Using
Selenoxide Elimination
Guoping Li,†,‡ Dongjian Zhu,†,‡ Qing Liu,†,‡ Lin Xue,*,† and Hua Jiang*,†
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of
Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing,
100190 P. R. China, and University of Chinese Academy of Sciences, Beijing, P. R. China
hjiang@iccas.ac.cn; zinclin@iccas.ac.cn
Received March 13, 2013
ABSTRACT
A new strategy for HOCl-specific fluorescent probes has been reported based on a selenoxide elimination reaction. Probes CM1 and CM2 were
synthesized as the first fluorescent probes containing an arylseleno moiety for hypochlorite according to this strategy. Both probes displayed
excellent properties, including high selectivity and sensitivity, fast response, and pH independency toward hypochlorite in vitro and vivo.
Reactive oxygen species (ROS) and reactive nitrogen
species (RNS) are well-known for their chemical reactivity
and tight connection with various physiological processes
and diseases.1 Hypochlorous acid (HOCl), one of the most
significant ROS, exerts a wide variety of physiological and
pathological effects in living systems.2 Endogenous HOCl,
which is produced from the reaction of hydrogen peroxide
with chloride catalyzed by myeloperoxidase (MPO), plays
an important role in protecting the body against invasion
of pathogens.3 Nevertheless, excessive generation of this
strong oxidant can also lead to many inflammation-related
diseases, including rheumatoid arthritis,4 cardiovascular
diseases,5 atherosclerosis,6 renal disease,7 lung injury,8 and
so on. Therefore, it is of vital importance to devise efficient
ways to selectively detect HOCl in biological systems so as
to understand its biological roles.
Fluorescence spectroscopy, which features real-time and
real-space detection, and nondestruction, has been regarded
as the most promising technique for detection of various
ROS/RNS.9 So far a number of small-molecule fluorescent
probes for specific detection of HOCl/OClÀ have been
† Beijing National Laboratory for Molecular Sciences.
‡ University of Chinese Academy of Sciences.
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10.1021/ol4006823
Published on Web 04/09/2013
2013 American Chemical Society

reported;10 however, most of them still face some draw-
backs, such as poor selectivity,10a,b,h,o low sensitivity,10d,e,m,o
and pH dependency.10c,e,g,k Therefore, novel fluorescent
probes that can overcome these limitations in detecting
HOCl/OClÀ are in urgent need.
or peroxynitrite have been reported recently,12 all of them
are based on a conventional PET (photoinduced electron
transfer) mechanism and their responsive rate,12a,cÀe pH
dependency,12b,e and water solubility12c still need further
refinement. In the present work, we reported the design,
synthesis, and photophyscial evaluation of the probes CM1
and CM2 in aqueous buffer and their cellular applications.
Herein, we have developed a new strategy for HOCl-
specific fluorescent probes based on a selenoxide elimina-
tion reaction that can well address the existing issues. The
selenoxide elimination was synthetically used to prepare R,
β-unsaturated esters and ketones, olefins and allyl alcohols
in mild conditions.11 As shown in Scheme 1a, the selen-
oxide elimination generally involves a two-step process.
The phenylselenenyl group is first oxidized by selected
oxidants, such as H2O2, leading to the formation of
intermediate selenoxide, which is then eliminated via a
spontaneous intramolecular syn elimination to generate a
conjugated CdC bond.11d We reason that utilizing this mech-
anism to design fluorescent probes based on R-arylseleno
carnonyl compounds will provide a new strategy for
selective detection of ROS. To verify our hypothesis, the
proof-of-concept fluorescent probes CM1 and CM2
(Scheme 1b) based on coumarin fluorophores were de-
signed. We envision that very high specificity for hypo-
chlorite could be achieved through a significant fluorescent
turn-on signal if the conjugated structures of probes CM1
and CM2 are recovered by selenoxide elimination in the
presence of the specific oxidant hypochlorite. Although
several selenium-based fluorescent probes for hypochlorite
Scheme 1. (a) General Process of Selenoxide Elimination;
(b) Synthesis and Proposed Fluorescent Turn-On Strategy
of the Probes
As shown in Scheme 1b, the probes can be easily synthe-
sized by reduction of commercially available coumarin dyes
and subsequent selenylation. The detailed synthetic proce-
dures were described in the Supporting Information. To
assess the success of our design concept, we first measured
the photophysical properties of CM1 and CM2 in PBS buffer
containing 10 mM phosphate, 8 g LÀ1 NaCl, 0.2 g LÀ1 KCl,
pH = 7.4. Both CM1 and CM2 display no major absorption
band and fluorescence emission (Φ < 0.001) in the range
of 300À600 nm, as the conjugated structures of probes are
broken by selenylation. With the addition of excess NaOCl
to the solutions, the absorption around 370À400 nm and
fluorescence centered at 480 nm (CM1, Φ = 0.036) and
468 nm (CM2, Φ = 0.047) characteristic of corresponding
coumarin dyes were observed (Figure S1).13 The phenom-
ena suggest that hypochlorite triggers the selenoxide elim-
ination so as to restore the conjugated system of the highly
fluorescent coumarins, which have been confirmed by
HRMS analysis (Figures S2 and S3). In order to evaluate
the sensing process of OClÀ, elaborate fluorimetric mea-
surements were performed. As expected, the fluorescence
of CM1 increased gradually with the addition of NaOCl.
Finally, the maximum fluorescence intensity was obtained
while the concentration of NaOCl is up to 28 μM(Figure1a).
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Org. Lett., Vol. 15, No. 8, 2013
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In particular, the fluorescence intensity showed good linear-
ity with the concentration of NaOCl in the low level range
(0À6.4 μM). The detection limit (LOD) is thus calculated to
be as low as 10 nM (S/N = 3, Figures 1b and S4), indicating
that with CM1 it is feasible to quantitatively determine
hypochlorite in biological environments. Similarly, CM2 also
responds to NaOCl but requires much larger amounts of
NaOCl to complete the conversion (Figure S5). This can be
well explained by the fact that the methyl group of CM2
partially restricts the conformational preference for the
intramolecular syn elimination (Scheme 1a). Moreover, it is
worth noting that CM1 canrapidlyrespondtoNaOClwithin
seconds (Figure 1c), making it a good candidate for real-time
detection of hypochlorite generated in situ.
of CM1 and CM2 to NaOCl over a wide range of 4À9
(Figure S7). Considering the pKa of HOCl is 7.6,10c,f CM1
and CM2 are responsive to both HOCl and OClÀ at a
wider pH range than the reported probes. Moreover, the
probes are stable in PBS buffer despite being exposed to air
for 10 h (Figure S8). These data clearly suggest that the
probes are capable of selectively detecting hypochlorite
without interference by other biological ROS and pH
variations.
Figure 2. Application of CM1 to the enzymatic MPO/H2O2/ClÀ
system. (a) Fluorescence changes as a function of time for the
reaction of 4 μM CM1 in the enzyme-catalytic systems (PBS, 40 μM
H2O2,1μgmLÀ1 MPO, pH = 7.4, 37 °C). (b) Fluorescence response
of CM1 in the absence (O) and presence (b) of MPO, λex = 405 nm,
λem = 480 nm.
Since hypochlorous acid is the myeloperoxidase (MPO)-
derived oxidant in living organisms, we further evaluated
the feasibility of CM1 for monitoring HOCl/OClÀ in an
MPO/H2O2/ClÀ enzymatic system. The fluorescence in-
tensity periodically increased after adding H2O2 (40 μM)
into CM1 (4 μM) solution in PBS buffer (10 mM phos-
phate, 8 g LÀ1 NaCl, 0.2 g LÀ1 KCl, pH = 7.4) containing
1 μg mLÀ1 myeloperoxidase at 37 °C (Figure 2), whereas
no obvious fluorescence response to H2O2 was observed in
the absence of MPO. Thus, the observed fluorescence
increase must be attributed to HOCl/OClÀ generated in
the enzymatic reaction.
Figure 1. (a) Fluorescence spectra of CM1 (4 μM) upon addition
of NaOCl (0À32 μM). (b) Fluorescence intensities of CM1 at
480 nm as a function of the concentrations of NaOCl in the
range of 0À6.4 μM. (c) Fluorescence response of CM1 (4 μM) to
NaOCl; arrows represent addition of 4 μM NaOCl. (d) Fluo-
rescence responses of CM1 and CM2 (4 μM) to NaOCl (100 μM)
and other ROS/RNS (200 μM). Bars represent emission inten-
sity ratios after (F) and before (F0) addition of each ROS/RNS.
With these data in hand, we next applied CM1 to living
cells for fluorescence imaging of OClÀ. After incubation with
5 μM CM1 at 37 °C for 20 min, NIH 3T3 cells exhibited faint
fluorescence in the optical window 420À520 nm (Figure 3a).
Treating the cells with 50 μM NaOCl for 30 s led to
remarkable fluorescence enhancement (Figure 3b), indicat-
ing that CM1 is extremely sensitive for detecting exogenous
OClÀ in living cells.
Inspired by these results as demonstrated above, we further
evaluated whether the probe CM1 could be used to monitor
endogenous hypochlorite in living cells. Because MPO is
abundantly expressed in primary azurophilic granules of
leukocytes including neutrophils14 and macrophages,15
RAW 264.7 macrophages and HL-60 human progranulocy-
tic leukemia cell lines were chosen as bioassay models for
endogenous OClÀ. After incubation with 5 μM CM1 for
Data were acquired at 25 °C in PBS buffer, λex = 405 nm, λem
480 nm for CM1, λem = 468 nm for CM2.
=
To evaluate whether the probes can selectively respond to
OClÀ under simulated physiological conditions, the fluor-
escence responses of the probes to other potentially compet-
ing ROS/RNS, which were prepared according to the
reported procedures (Supporting Information), were also
performed. As shown in the selectivity profiles (Figure 1d),
only OClÀ incurs a dramatic fluorescence enhancement for
CM1 and CM2, respectively. Other ROS or RNS, includ-
ing ONOOÀ, H2O2, t-BuOOH (TBHP), HO•, t-BuOO•
(ROO•), 1O2, O2À, NO2À, NO3À, and NO, exert no obvious
spectral changes. Moreover, other biological thiols, which
are abundant in living samples, have no effect as well under
identical conditions (Figure S6).
(14) Hampton, M. B.; Kettle, A. J.; Winterbourn, C. C. Blood 1998,
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Additionally, we also evaluated the effect of pH on the
detection of hypochlorite. The variations of pH cause no or
little influence on the fluorescence intensity and response
2004
Org. Lett., Vol. 15, No. 8, 2013

much stronger fluorescence was detected (Figure 4c). Now
that CM1 is highly selective for hypochlorite over other
ROS/RNS (Figure 1d), it is reasonable to believe that the
exogenous H2O2 activated myeloperoxidase in HL-60 cells
to transfer ClÀ to OClÀ and consequently caused the great
enhancement of fluorescence.10b Lipopolysaccharide (LPS),
an endotoxin, is commonly used for eliciting strong immune
responses in mammalian cells and producing highly active
ROS.16 We further incubated the RAW 264.7 macrophage
cells with LPS for 16 h (12 h first, and then additional 4 h) to
produce endogenous OClÀ and then stained the cells with
5 μM CM1 for 20 min.10k Under these conditions, the
pseudocolor images also displayed a sharp contrast to that
of the control (Figure 4e and g), suggesting that CM1 had
successfully detected OClÀ generated by the stimulation of
LPS. Therefore, we can conclude that CM1 is capable of
monitoring and imaging both exogenous and endogenous
HOCl/OClÀ with high sensitivity and selectivity in living cells.
In summary, on the basis of a specific intramolecular
selenoxide elimination reaction, we have demonstrated a
novel strategy in designing fluorescent probes for the
highly selective detection of hypochlorite. The probe,
CM1, which features high sensitivity, selectivity, fast re-
sponse, and pH independency, was successfully utilized in
detecting hypochlorite in aqueous media and living cells.
Importantly, it can be used to visualize the generation of
endogenous hypochlorite in progranulocytes and macro-
phages. We emphasize that this novel design strategy,
which is easily adaptable, will provide a general method
to construct HOCl-specific probes from various commer-
cially available dyes with additional tunable photophysical
properties. The development of ratiometric fluorescent
probes based on this strategy is also a challenging project
that is underway.
Figure 3. Fluorescent imaging of exogenous OClÀ in CM1-
labeled NIH 3T3 cells. (a) Cells were stained with 5 μM CM1
at 37 °C for 20 min. (b) CM1-labeled cells were treated with
50 μM NaOCl for 30 s at 25 °C. (c) Bright-field image of (a) and
(b). Emission intensities were collected in an optical window
420À520 nm, λex = 405 nm, Scale bar: 10 μm.
Figure 4. Fluorescent imaging of endogenous OClÀ in CM1-
labeled HL-60 (top) and RAW 264.7 cells (bottom). (a) Fluo-
rescence images of HL-60 cells loaded with 5 μM CM1 at 37 °C
for 30 min. (b) Bright-field images of cells in panel (a). (c) The
cells after addition of 100 μM H2O2 at 25 °C for 20 min. (d)
Bright-field images of cells in panel (c). (e) Fluorescence images
of RAW 264.7 cells loaded with 5 μM CM1 at 37 °C for 20 min.
(f) Bright-field images of cells in panel (e). (g) After pretreatment
with 3 μg mLÀ1 LPS for 12 h and additional 1 μg mLÀ1 LPS for
4 h, the cells loaded with 5 μM CM1 at 37 °C for 20 min. (h)
Bright-field images of cells in panel (g). Emission intensities were
collected in an optical window 420À520 nm, λex = 405 nm.
Intensity bar: 0À500.
Acknowledgment. We thank the National Natural Science
Foundation of China (21102148, 21125205, 212210017),
National Basic Research Program of China (2009CB930802,
2011CB935800), and the State Key Laboratory of Fine
Chemicals, Department of Chemical Engineering, Dalian
University of Technology for financial support.
20 min at 37 °C, respectively, both kinds of cells exhibited
very weak fluorescence (Figure 4a and e). When the CM1-
loaded HL-60 cells were treated with 100 μMH2O2 for 20 min,
Supporting Information Available. Synthetic proce-
dures, characterization of compounds, and additional
spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Yotsumoto, S.; Aratani, Y.; Ohno, N.; Petty, H. R. J. Immunol. 2006,
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The authors declare no competing financial interest.
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