HKOCl-2 series of green BODIPY-based fluorescent probes for hypochlorous acid detection and imaging in live cells

Article abstract of DOI:10.1021/ol501496n

A HKOCl-2 series of new fluorescent probes for hypochlorous acid (HOCl) detection in live cells is reported. The probes exhibit excellent selectivity, sensitivity, and chemostability toward HOCl. In particular, HKOCl-2b rapidly and selectively detects endogenous HOCl in both human and mouse macrophages. These probes could therefore serve as promising discovery tools to help elucidate biological functions of HOCl.

Full text of DOI:10.1021/ol501496n

Letter
pubs.acs.org/OrgLett
HKOCl‑2 Series of Green BODIPY-Based Fluorescent Probes for
Hypochlorous Acid Detection and Imaging in Live Cells
Jun Jacob Hu,† Nai-Kei Wong,† Qiangshuai Gu, Xiaoyu Bai, Sen Ye, and Dan Yang*
Morningside Laboratory for Chemical Biology, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong
Kong, P. R. China
S
* Supporting Information
ABSTRACT: A HKOCl-2 series of new fluorescent probes for
hypochlorous acid (HOCl) detection in live cells is reported. The probes
exhibit excellent selectivity, sensitivity, and chemostability toward HOCl.
In particular, HKOCl-2b rapidly and selectively detects endogenous HOCl
in both human and mouse macrophages. These probes could therefore
serve as promising discovery tools to help elucidate biological functions of
HOCl.
eactive oxygen/nitrogen species (ROS/RNS) such as
designed HKOCl-2a with two methyl groups added to the 1
Rsuperoxide, hydrogen peroxide, nitric oxide, hydroxyl and 7 positions of the BODIPY core of HKOCl-1 to increase
the steric hindrance around 4-methoxyphenol (Scheme 1). One
radical, hypochlorous acid (HOCl), and peroxynitrite are
involved in fundamental cellular processes including inflamma-
tion, aging, cancer, and neurodegeneration.1 Over the past
decade, extensive efforts have been invested in the development
of selective fluorescent probes for imaging cellular ROS/
RNS.2,3 Among ROS/RNS, HOCl is a highly potent oxidant
and exhibits remarkable reactivity toward various protein side
chains and peptide bonds,4 which in part accounts for its role as
an important microbicidal effector in innate immunity.5−7
Endogenous HOCl can be generated in a regulated manner
from hydrogen peroxide and chloride by the enzyme
myeloperoxidase (MPO).8 However, abnormal HOCl produc-
tion derived from phagocytes is linked to many diseases
including cardiovascular diseases,9,10 inflammatory diseases,4,11
neurodegenerative diseases,12 cystic fibrosis,13 kidney dis-
eases,14 and certain cancers.15
Although many fluorescent probes for HOCl detection have
been reported in recent years,16−46 only a fraction of them
showed practical utility in further biological investigations due
to common problems in selectivity, sensitivity, chemostability,
and/or photostability.
Previously, our group reported a green fluorescent probe
HKOCl-1 for HOCl with high selectivity and sensitivity.21
However, its fluorescent product A can be further oxidized by
HOCl to give compounds of weaker fluorescence, resulting in
compromised fluorescence enhancement. One possible decom-
position pathway is the epoxidation of the quinone moiety, as
we found that 4-methoxyphenol was indeed oxidized by 2 equiv
of HOCl to give 2,3-epoxy-1,4-benzoquinone in 65% yield (see
the Supporting Information (SI)). To overcome this limitation,
we have developed the HKOCl-2 series of fluorescent probes
with excellent selectivity, sensitivity, and chemostability for
HOCl detection and imaging in live cells.
Scheme 1. Structures and Design of HKOCl-1 and an
HKOCl-2 Series of New Fluorescent Probes for HOCl
Detection
additional advantage is that the quantum yield of the
fluorescent product B is expected to be higher than that of A
because the rotational degree of freedom of the quinone moiety
is further restricted by the two added methyl groups.47
Moreover, ortho-halogen substituted analogues HKOCl-2b
and HKOCl-2c have been designed to further block oxidation
of the quinone products C and D, which have been confirmed
by ESI-MS measurement (see the SI). Furthermore, according
to the HOMO energy calculations (Scheme S1), the extra
substituents on HKOCl-2a−c should not change the
mechanism for fluorescence turn-on upon oxidation by
HOCl, that is, HKOCl-2a−c are expected to be nonfluorescent
due to the photoelectron transfer (PeT) process whereas
oxidation products B−D should be fluorescent due to
suppression of PeT.
The designed HKOCl-2 series of probes (HKOCl-2a,
HKOCl-2b, and HKOCl-2c) has been prepared from
In order to prevent the overoxidation of the quinone product
A, generated by HKOCl-1 reaction with HOCl, we have
Received: May 23, 2014
Published: June 20, 2014
© 2014 American Chemical Society
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Figure 1. Fluorescence emission spectra of the fluorescent probes (A) HKOCl-2a (10 μM), (B) HKOCl-2b (10 μM), and (C) HKOCl-2c (10 μM)
in 0.1 M phosphate buffer (0.1% DMF, pH 7.4) upon treatment of increasing amounts (0−3 equiv) of HOCl for 30 min.
corresponding aldehydes and methylated pyrrole according to
Scheme S3. We first investigated the reactivity of those probes
toward HOCl in 0.1 M potassium phosphate buffer at pH 7.4.
As expected, in the absence of HOCl, the probes were
nonfluorescent; on exposure to HOCl, a very rapid
fluorescence increase was observed with emission maxima at
around 545 nm (Figure 1), and the fluorescence intensity
reached a plateau within 15 min (Figure 2).
linear, the detection limit (S/N = 3) was estimated to be 42, 18,
and 37 nM for HKOCl-2a, HKOCl-2b, and HKOCl-2c,
respectively. Overall, the above-mentioned results confirmed
the excellent chemostability of the fluorescent products of the
HKOCl-2 series of fluorescent probes upon treatment with
HOCl.
Next, the selectivity of the HKOCl-2 series of fluorescent
probes toward various analytes has been tested. As shown in
Figure 4, over 900-fold enhancement in fluorescence intensity
was observed with HKOCl-2b (10 μM) in the presence of 2
equiv of HOCl (final concentration 20 μM), whereas 10 equiv
1
•
of other ROS/RNS including H2O2, O2, ROO•, TBHP, NO,
O2
,
•OH, and ONOO− (2 equiv) induced negligible
•−
fluorescence increase after 30 min (Figure 4). HKOCl-2a and
HKOCl-2c have also been found to be highly selective toward
HOCl (Figure 4). According to literature, under physiological
or pathophysiological conditions, the steady-state ONOO−
concentrations are estimated to be in the nanomolar range,48
while concentrations of HOCl are estimated to be in the
micromolar range.49 As such, the peroxynitrite concentration is
unlikely to surpass that of HOCl in most cases, to become an
important interfering species in HOCl detection. Collectively,
the excellent selectivity of the HKOCl-2 series of fluorescent
probes, especially HKOCl-2b, toward HOCl over other ROS/
RNS, confirms our design principle.
Figure 2. Time course of fluorescence intensity of probes HKOCl-
2a−c upon treatment with 2 equiv of HOCl over a period of 30 min.
Probes were dissolved in 0.1 M potassium phosphate buffer at pH 7.4
to a final concentration of 10 μM (containing 0.1% DMF).
As shown in Figure 3, the fluorescence intensity of all three
As pH is an important factor in cellular HOCl detection, the
pH-dependence of our probe HKOCl-2b was validated in
buffers prepared in the biologically relevant pH range (3−12).
The fluorescence increase of HKOCl-2b toward HOCl was
significant across a wide range of pH (4−10; Figure 5). Thus,
this result not only demonstrates the excellent pH stability of
HKOCl-2b but also illustrates that our probe HKOCl-2b is
likely to be biocompatible.
probes at around 550 nm became saturated when 2 equiv of
Based on its superior performance in selectivity and
sensitivity in chemical characterization, HKOCl-2b was chosen
among the three probes for functional elaboration in live cell
imaging. To induce endogenous HOCl, RAW264.7 mouse
macrophages were coincubated with HKOCl-2b (10 μM) and
the PKC activator PMA (phorbol myristate acetate; 200 ng/
mL) or Toll-like receptor-2 (TLR2) ligand zymosan (Saccha-
romyces cerevisiae; 10 μg/mL) for 30 min, followed by confocal
imaging (Figure 6). The NOX (NADPH oxidase) specific
peptide inhibitor gp91ds-tat (2.5 μM) was added to block
superoxide production. As anticipated, both PMA and zymosan
significantly increased HOCl production in activated macro-
phages (Figure 6), with a very well-defined cytoplasmic
fluorescence distribution. Inhibition of superoxide generation
by gp91ds-tat potently reduced the HKOCl-2b fluorescence
signal to near basal level, confirming that the observed ROS
Figure 3. Dose response of fluorescence emission intensity of probes
HKOCl-2a−c toward HOCl (0−3 equiv). Probes were dissolved in
0.1 M potassium phosphate buffer at pH 7.4 to a final concentration of
10 μM (containing 0.1% DMF). The fluorescence spectra were
recorded at 30 min after addition of HOCl.
HOCl were added and almost no change in fluorescence
intensity was observed upon treatment with 10 equiv of HOCl
(Figure S2), demonstrating the chemostability of fluorescent
products toward HOCl. Among the three probes (Φ < 0.002),
HKOCl-2b gave the strongest turn-on fluorescence response
toward HOCl (Figure 3). Although the dose response was not
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Figure 4. Test of selectivity of fluorescent probes (A) HKOCl-2a (10 μM), (B) HKOCl-2b (10 μM), and (C) HKOCl-2c (10 μM) in 0.1 M
phosphate buffer (0.1% DMF, pH 7.4) toward various ROS and RNS. Fluorescence spectra were recorded at 30 min.
Figure 7. Detection of endogenous HOCl in differentiated THP-1
human macrophages. (A) Cells were incubated with HKOCl-2b (10
μM) in the presence or absence of PMA (200 ng/mL), PMA plus
Go6983 (100 nM), or PMA plus DPI (100 nM) for 30 min, before
̈
Figure 5. Fluorescence response of HKOCl-2b (10 μM) in 0.1 M
potassium phosphate buffer across a wide pH range (3−12) toward
HOCl (20 μM) was measured.
confocal imaging. (B) Quantitative analysis of endogenous HOCl
production by measuring the mean fluorescence of cells under the
same pharmacological treatments as in (A) (n = 6−13 cells). ***, p <
0.001. Scale bar represents 10 μm.
reproducibly manipulated by established pharmacological
agents.
Lastly, to further validate the selectivity of HKOCl-2b
toward HOCl in cells, we used yeast zymosan as a stimulant to
mimic the phagocyte oxidative burst in response to fungal
infection. Differentiated THP-1 cells were coincubated with
HKOCl-2b (10 μM) and zymosan (10 μg/mL) in the presence
or absence of the specific HOCl scavenger taurine (5 mM) or
MPO inhibitor ABAH (4-aminobenzoic acid hydrazide; 100
μM) for 30 min, followed by confocal imaging (Figure 8).
Zymosan rapidly triggered HKOCl-2b fluorescence enhance-
ment comparable to that in PMA treatment. As expected,
taurine and ABAH greatly attenuated the HKOCl-2b
fluorescence signal to basal levels, again suggesting the high
selectivity of the probe toward HOCl.
Figure 6. Detection of endogenous HOCl in activated RAW264.7
mouse macrophages. (A) Cells were incubated with HKOCl-2b (10
μM) in the presence or absence of PMA (200 ng/mL), zymosan (10
μg/mL), or zymosan plus gp91ds-tat (2.5 μM) for 30 min, before
confocal imaging. (B) Quantitative analysis of endogenous HOCl
production by measuring the mean fluorescence of cells under the
same pharmacological treatments as in (A) (n = 20 cells). ***, p <
0.001. Scale bar represents 10 μm.
production was enzymatically controlled. The results suggest
that HKOCl-2b is highly sensitive and rapid in its turn-on
response in live cells.
Next, we attempted to visualize endogenous HOCl in human
THP-1 monocytic macrophages, which have not been validated
for HOCl production despite being known to express MPO at
low to moderate levels.50 THP-1 cells were first differentiated
into macrophages as previously described.51 Cells were then
coincubated with HKOCl-2b (10 μM) and PMA (200 ng/mL)
in the presence or absence of the PKC inhibitor Go6983 (100
̈
Figure 8. Test on selectivity of HKOCl-2b toward endogenous HOCl
in differentiated THP-1 human macrophages. (A) Cells were
incubated with HKOCl-2b (10 μM) in the presence or absence of
zymosan (10 μg/mL), zymosan plus taurine (5 mM), or zymosan plus
ABAH (100 μM) for 30 min, before confocal imaging. (B)
Quantitative analysis of endogenous HOCl production by measuring
the mean fluorescence of cells under the same pharmacological
treatments as in (A) (n = 6−13 cells). ***, p < 0.001. Scale bar
represents 10 μm.
nM) or NOX inhibitor DPI (100 nM) for 30 min, followed by
confocal imaging (Figure 7). In the presence of PMA, the
HKOCl-2b fluorescence response was significantly enhanced,
while both Go6983 and DPI could efficiently reduce this
̈
increased fluorescence to a level comparable to that of the
unstimulated control. These results suggest that endogenous
HOCl production in differentiated human macrophages is
coupled to the PKC/NOX signaling pathway and can be
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, characterization, and experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: yangdan@hku.hk.
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Author Contributions
†J.J.H. and N.-K.W. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by The University of Hong Kong, the
University Development Fund, and Hong Kong Research
Grants Council under General Research Fund Scheme (HKU
706410). We thank Prof. Heping Cheng for very helpful
discussions, Dr. Yan Chen and Dr. Jing Guo for technical
assistance, and the HKU Li Ka Shing Faculty of Medicine Core
Facility for support in confocal microscopy.
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