Development of a selenide-based fluorescent probe for imaging hypochlorous acid in lysosomes

Article abstract of DOI:10.1016/j.jphotochem.2014.10.015

The development of fluorescent probes for hypochlorous acid (HOCl) has received intense attention because of the biological significance of HOCl. In this work, a novel fluorescent probe based on a selenide switch for the detection of HOCl in lysosomes has been designed and synthesized on a 1,8-naphthalimide scaffold. The probe exhibited a high selectivity for HOCl over various reactive oxygen species (ROS) with a fast response and a large fluorescence enhancement in aqueous media. Confocal microscopy imaging of living cells indicated that the probe was able to accumulate in lysosomes and was successfully applied to imaging exogenous HOCl in living cells. Attempts of using Lyso-NI-Se to image HOCl in stimulated RAW264.7 cells failed, probably due to the absence of endogenous HOCl in lysosomes or the undesirable detection limit.

Full text of DOI:10.1016/j.jphotochem.2014.10.015

Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Development of a selenide-based fluorescent probe for imaging
hypochlorous acid in lysosomes
a
a
b
a,
Zongjin Qu , Junxia Ding , Meiyu Zhao , Peng Li *
a
State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Institute of Theoretical Simulation Chemistry, Academy of Fundamental and Interdisciplinary Science, Harbin Institute of Technology, Harbin 150080, China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 7 July 2014
Received in revised form 15 October 2014
Accepted 20 October 2014
Available online 22 October 2014
The development of fluorescent probes for hypochlorous acid (HOCl) has received intense attention
because of the biological significance of HOCl. In this work, a novel fluorescent probe based on a selenide
switch for the detection of HOCl in lysosomes has been designed and synthesized on a 1,8-naphthalimide
scaffold. The probe exhibited a high selectivity for HOCl over various reactive oxygen species (ROS) with a
fast response and a large fluorescence enhancement in aqueous media. Confocal microscopy imaging of
living cells indicated that the probe was able to accumulate in lysosomes and was successfully applied to
imaging exogenous HOCl in living cells. Attempts of using Lyso-NI-Se to image HOCl in stimulated
RAW264.7 cells failed, probably due to the absence of endogenous HOCl in lysosomes or the undesirable
detection limit.
Keywords:
Fluorescent probes
Naphthalimide
Lysosomes
ã 2014 Elsevier B.V. All rights reserved.
Selenium
Hypochlorous acid
1. Introduction
monitoring cellular HOCl under fluorescent microscopy [19–37].
However, there are still opportunities to improve these probes in
Due to their chemical reactivity, reactive oxygen species (ROS)
and reactive nitrogen species (RNS) are interrelated in innumera-
ble ways with various physiological processes and health problems
terms of selectivity, sensitivity, response speed and subcellular
localization. Recently, a novel nanodosimeter for HOCl with
lysosome-homing specificity was reported and applied to imaging
exogenous HOCl [33]. However, the area of small molecule HOCl-
probes preferentially directed to lysosomes has not been explored.
In this work, we present Lyso-NI-Se, a lysosome-targetable
fluorescent probe for imaging HOCl in living cells. The optical
probe was designed and synthesized on a naphthalimide platform
(Scheme 1). Besides the fluorophore, the structural key points of
the probe are the selenide group and the (aminoethyl) morpholine
moiety. Previous work has established that selenide could be
selectively oxidized by HOCl [35–37], so we chose selenide as the
fluorescent modulator. On the other hand, the morpholine moiety
was expected to serve a function of leading this probe to lysosomes
[38–40]. The probe was synthesised by a nucleophile substitution
reaction between terminal bromine and aniline in the presence of
KOH with CuI as the catalyst to afford a yield of 73%, and was well
[1–6]. Hypochlorous acid (HOCl), an important member of ROS,
plays essential roles in inflammation and the immune defence
against microorganisms [7]. Nevertheless, persuasive evidences
have shown that excessive or misplaced HOCl is implicated in
various human diseases, such as neurodegeneration, cardiovascu-
lar diseases, arthritis and even cancers [8–12]. Besides, production
of this highly reactive and powerful two-electron oxidizing agent
can lead to downstream generation of RNS and other ROS which
will cause cell injury by nitration, chlorination or oxidation of
biomolecules [9]. Endogenous HOCl is produced by myeloperox-
2 2
idase (MPO) from chloride and H O within phagosomes [13–16].
In particular, HOCl could induce apoptosis of cultured cells through
the rupture of lysosomes [17]; not only locales where ROS are
generated, but also lysosomes play important roles in regulating
ROS flux, which determines the ultimate biological outcome of ROS
as well as the cross-talk between subcellular compartments [18].
In this regard, it is necessary to devise efficient ways to detect HOCl
in lysosomes of living cells.
1
13
77
characterized by H NMR, C NMR, Se NMR and HRMS.
2. Experimental
Fluorescent probes are powerful tools in cell biology and a
variety of HOCl-sensitive probes have been developed for
2.1. Materials
Unless otherwise noted, all chemicals were analytical grade
from commercial suppliers and used without further purification.
The stock solution of the probe Lyso-NI-Se (acetonitrile or DMSO,
*
Corresponding author. Tel.: +86 411 84379293; fax: +86 411 84675584.
E-mail address: pengl@dicp.ac.cn (P. Li).
ꢀ
1.0 mM) could be maintained in refrigerator at 4 C for more than
http://dx.doi.org/10.1016/j.jphotochem.2014.10.015
010-6030/ã 2014 Elsevier B.V. All rights reserved.
1

2
Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
Scheme 1. Synthetic procedures of Lyso-NI-Se and the reaction scheme for the detection of HOCl.
2
months. Ultrapure water was used throughout. Hypochlorite acid
NMR spectrometer. For 1H and 13C NMR, TMS in the solvents
7
7
(
HOCl), tert-butylhydroperoxide (t-BuOOH) and hydrogen perox-
indicated was internal reference; for Se NMR, external reference
PhSeSePh was used. Multiplicities of signals are described as
follows: s — singlet, br — broad, d — doublet, t — triplet, m —
multiplet. High resolution mass spectra were obtained by an
Agilent Q-TOF 6540 spectrometer. Element analyze was carried out
on vario EL III (Elementar). Florescent images were taken by
FV1000 confocal laser-scanning microscope (Olympus).
ide (H ) stock solutions were diluted by water from commercial
NaClO solution, t-BuOOH (70%) and H
Superoxide anion (O
dry DMSO. Hydroxyl radical (HO ) was produced in situ by Fenton
reaction between H O (200 mM) and FeCl (100 mM), and the
2 2 2
concentration of HO was represented by the concentration of Fe
Peroxynitrite (ONOO ) was prepared from nitrosation of H
2 2
O
2
O
2
(30%), respectively.
À
2
) was delivered by adding KO
2
dissolved in
ꢁ
ꢁ
2+
.
À
2
O
2
by
isoamyl nitrite [41]. NO was generated from sodium nitroprusside
2.3. Absorption and fluorescence analysis
À
(
SNP). The concentrations of ONOO and NaClO were ascertained
À1
À1
by absorbance spectra
e
(e
NaClO,292 nm = 350 M cm
and
Absorption and fluorescence spectra were recorded at room
temperature with 10-mm glass cells. The probe Lyso-NI-Se
(acetonitrile, 1.0 mM) was diluted by acetonitrile and aqueous
ONOOÀ
À1
À1
,302 nm = 1670 M cm ) just before use. DMSO solutions
of Lyso-NI-Se (1 mM) and neutral red (NR, 1 mM) were used for
cell-staining. Phosphate buffer saline (PBS) was aqueous solution
containing phosphate (10 mM), NaCl (8.0 g/L) and KCl (0.2 g/L), pH
buffer. Typically, Lyso-NI-Se (acetonitrile, 50
trile (4950 L) and phosphate buffer (PB, 5000
were added into a 10-mL glass comparison tube to get a probe
solution (5.0 M); then, NaClO (10 L, 5.0 mM) was added and the
m
L, 1.0 mM), acetoni-
m
m
L, 0.1 M, pH 5.0)
7.4.
m
m
2.2. Instruments
absorption or fluorescence spectra were measured immediately.
Steady-state UV/vis spectra were measured on a Lambda 35
2.4. Cell culture
UV–vis spectrophotometer (PerkinElmer). Fluorescence spectra
were obtained from Fluoromax-4 spectrofluorometer (Horiba-
Jobin Yvon, monochromator calibrated) with the widths of the
MCF-7 or RAW264.7 cells (ATCC, USA) were maintained
following protocols from the American Type Culture Collection.
1
13
6
À1
excitation and emission slits 5 nm and 4 nm, respectively. H, C,
Cells were subcultured at a density of 1 Â10 cells mL by seeding
7
7
and Se NMR spectra were taken on a Bruker 400 MHz or 500 MHz
on 35 mm  12 mm glass bottom cell culture dishes and cultured in
Fig.1. Fluorescence (a) and absorption (b) spectra of Lyso-NI-Se (5.0
m
M) treated with NaClO (HOCl source, 0–8.0
mM). (c) GSH can reduce the signal (the arrow represents the
addition of 123 M GSH). Data were acquired in 1:1CH CN–H O solutions at pH 5.0 (50 mM phosphate buffer) with l
m
3
2
ex = 430 nm.

Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
3
7
3
cells were incubated with NR (5
0 min in PBS at 37 C and washed 3 times with PBS before taking
m
M) and Lyso-NI-Se (5
m
M) for
ꢀ
images; although Lyso-NI-Se was weakly fluorescent, palpable
discrete fluorescence patterns could be acquired with proper
instrument settings. Fluorescent images were processed and
analyzed with FV10-ASW 3.0 Viewer software.
2
.6. Synthesis and characterization of compounds
2
.6.1. Synthesis of N-(morpholinoethylamino)-4-bromo-1,8-
naphthalimide
A
mixture of 4-bromo-1,8-naphthalic anhydride (2.5 g,
0.009 mol), 4-(2-aminoethyl)-morpholine (2.5 g, 0.018 mol) in
5
0 mL ethanol were refluxed for 8 h. Then the reaction mixture
was evaporated on a rotary evaporator until dry and purified on
silica gel chromatography (200–300 mesh) eluted with dichloro-
methane: ethyl acetate = 1:1 (V/V) to give the product as white
1
solid (2.5 g, 72.5%). H NMR (400 MHz, CDCl
3
)
d
8.63 (d, J = 7.6 Hz,
1
1
4
1
1
H), 8.54 (d, J = 8.4 Hz, H), 8.38 (d, J = 7.6 Hz, H), 8.02 (d, J = 7.6 Hz,
H), 7.83 (t, J = 8.0 Hz, H), 4.33 (t, J = 6.8 Hz, H), 3.68 (t, J = 8.4 Hz,
H), 2.71 (t, J = 6.8 Hz, H), 2.60 (br, H). C NMR (CDCl
ppm): 163.58,163.56,133.25,132.00,131.19, 131.10, 130.64, 130.26,
29.02, 128.07, 123.08, 122.21, 67.02, 56.09, 53.82, 37.33 [40].
1
2
2
4
13
3
,100 MHz) d
(
1
2
.6.2. Synthesis of (4-nitrophenyl)(phenyl) selane
Under N in ice–water bath, diphenyl diselenide (5.07 g,
6.2 mmol) was reduced by NaBH (1.52 g, 40.2 mmol) in ethanol
100 mL) to get a colorless solution. Then p-nitrochlorobenzene
5.12 g, 32.5 mmol) was added and the reaction mixture was
2
1
(
(
4
Fig. 2. Calibration curve for detecting HOCl. Working conditions: 10.0
Se, pH 5.0, CH CN/H O = 20/1, AcOH–AcONa buffer (final concentration, 5 mM),
em = 425/530 nm.
m
M Lyso-NI-
refluxed for 4 h. The solvent was evaporated and the residue was
purified on silica gel chromatography (200–300 mesh) eluted with
3
2
l
ex/
petroleum ether: ethyl acetate = 50:1 (V/V) to give an orange solid
ꢀ
(
6.70 g, 74%). m.p. 64–66 C. The product was used directly for the
RPMI1640 containing 10% fetal bovine serum supplemented with
next step [42].
ꢀ
penicillin (100 U/mL) and streptomycin (100
m
g/mL) at 37 C for
2
4 h in a humidified atmosphere containing 5% CO
.5. Confocal fluorescence imaging
Florescent images were taken by FV1000 confocal laser-
2
.
2
.6.3. Synthesis of 4-(phenylselanyl) aniline
A 100 ml stainless autoclave was charged with (4-nitro-
2
phenyl)(phenyl) selane (7.50 g, 27.0 mmol), Raney Ni (1.36 g) and
ethanol (50 ml). The reactor was purged with hydrogen for
3
for 15 h at 100 C [38]. Then the solvent was evaporated and the
residue was purified on silica gel chromatography
times, pressurized to 4.0 MPa with hydrogen, and maintained
scanning microscope (Olympus) with Â40 or Â100 objective
lenses. For the detection of exogenous and endogenous HOCl in
RAW264.7 cells, fluorescent images were taken using identical
optical instrument settings. For co-localization, RAW264.7 or MCF-
ꢀ
(
200–300 mesh) eluted with petroleum ether: ethyl acetate =
Fig. 3. (a) Fluorescence responses of Lyso-NI-Se to various ROS/RNS (bars represent enhancement factors at 540 nm): 1, free; 2, H
2
O
2
(200
M). (b) Lyso-NI-Se (5.0
CN–H O solutions at pH 5.0 (50 mM phosphate
m
M); 3, t-BuOOH (200
mM); 4,
À
À
ꢁ
À
À
OONO (100
reactionwith NaClO instantly. NaClO (final 6.3
buffer) with ex = 430 nm.
mM); 5, O
2
(200
m
M); 6, OH (100
m
M); 7, NO (200
m
M); 8, NaClO (6.3
m
M); 9, NO
3
(300.0
m
M); 10, NO
2
(300.0
m
mM) can finish the
m
M) was added at the time indicated by the arrow. Data were acquired in 1:1CH
3
2
l

4
Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
1
C
30.21, 129.06, 126.07, 116.56, 116.07. MS (API-ES): Calcd for
12
H11NSe 249.0, found [MH ] 250.1 [42].
+
2
.6.4. Synthesis of Lyso-NI-Se
CuI (0.21 g, 1.1 mmol), KOH (0.62 g, 11.1 mmol), N-(morpholi-
noethylamino)-4-bromo-1, 8-naphthalimide (0.94 g, 2.4 mmol)
and 4-(phenylselanyl) aniline (0.60 g, 2.4 mmol) were dissolved
ꢀ
in dry DMSO (20 mL) and stirred at 90 C under N
2
for 6 h. After
cooling to room temperature, the black sediments in the mixture
were removed by filtration. Then the solution was dried by
evaporation on a rotary evaporator and the residue was purified on
silica gel chromatography (200–300 mesh) eluted with dichloro-
methane: ethyl acetate = 3:1 (V/V) to afford Lyso-NI-Se as a red
1
6
solid (0.98 g, 73%). H NMR (500 MHz, DMSO-D )
d (ppm): 9.41 (s,
1
1
1
1
H), 8.76 (d, H, J = 8.5 Hz), 8.47 (d, H, J = 7.5 Hz), 8.27 (d, H,
1
2
J = 8.5 Hz), 7.78 (t, H, J = 7.75 Hz), 7.56–7.54 (m, H), 7.46–7.44 (m,
2
2
4
H), 7.39–7.31 (m, 6H), 4.15 (t, H, J = 6.75 Hz), 3.54–3.53 (m, H),
2
4
13
6
Fig. 4. The effect of pH on the fluorescence intensity of Lyso-NI-Se. Data were
2.55 (t, H, J = 7.0 Hz), 2.46 (s, H). C NMR (DMSO-D , 126 MHz)
ppm): 163.52, 162.68, 146.81, 140.73, 134.94, 133.11, 131.64, 131.39,
30.99, 129.55, 129.34, 128.97, 127.20, 125.21, 123.23, 122.62,
22.01, 121.97, 111.48, 108.84, 66.18, 55.65, 53.37, 36.48. Se NMR
DMSO-D , 95 MHz)
d
collected in 1:1CH
addition of NaClO (5.0
3
CN À H
2
O solutions with various pH values before after the
M).
(
1
m
M) to Lyso-NI-Se (5.0
m
7
7
1
(
5
:1 (V/V) to give a white solid (6.02 g, 94.0%) [39]. 1H NMR
400 MHz, CDCl (ppm): 7.39 (d, J = 8.0 Hz, 2H), 7.29 (d,
J = 7.2 Hz, 2H), 7.20–7.12 (m, 3H), 6.62 (d, J = 8.0 Hz, 2H), 3.71 (br,
6
d
(ppm): 402.49. Anal. Calcd for
(
3
) d
C
4
30 27 3 3
H N O Se: C, 64.75; H, 4.89; N, 7.55. Found: C, 64.03; H,
.67; N, 7.41. HRMS (ESI) Calcd for C30H N O Se [MH ] 558.1296,
28 3 3
+
13
3
2H). C NMR (CDCl , 100 MHz) d (ppm): 146.80, 137.10, 134.10,
found 558.1303.
Fig. 5. Lyso-NI-Se accumulates in lysosomes of RAW264.7 cells. (a) Fluorescence image from Lyso-NI-Se (channel 1,
from NR (channel 2, ex = 543 nm, em = 590–630 nm). (c) Bright field image. (d) Overlay of channels 1 and 2. (e) Intensity profile of ROI in (d). (f) Overlay of (a), (b) and the
bright field image. (For interpretation of the reference to color in the text, the reader is referred to the web version of this article.)
lex = 405 nm, lem = 500–550 nm). (b) Fluorescence image
l
l

Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
5
Fig. 6. Confocal fluorescent images of exogenous HOCl in RAW264.7 cells (
l
ex = 405 nm,
l
em = 500–550 nm) and the corresponding bright field images. (a) Cells were
ꢀ
incubated with 5
m
M Lyso-NI-Se for 30 min at 37 C and washed 3 times before imaging. (b) Cells in (a) were then treated with 5
m
M NaClO and pictured within 1 min.
2
.6.5. Synthesis of Lyso-NI-SeO
NaClO (4.5 mM, 50 mL) was added to Lyso-NI-Se (0.12 g)
dissolved in MeCN (600 mL) and the solution was stirred for
min at room temperature. Then the solution was dried by
540 nm (Fig. 1a), and the fluorescence enhancement was 22-fold in
the presence of 8.0 M NaClO. Meanwhile, the titration of Lyso-NI-
m
Se with NaClO did not change the featureless intramolecular
charge transfer (ICT) absorption band (Fig. 1b), which suggested
that the “turn-on” result might originate from inhibition of the
photoinduced electron transfer (PET) process from the selenide
pendant to the naphthalimide fluorophore. We also isolated the
1
evaporation on a rotary evaporator and the residue was purified on
silica gel chromatography (200–300 mesh) eluted with dichloro-
methane: methanol = 40:1 (V/V) to provide pure Lyso-NI-SeO as a
1
1
13
red solid (0.08 g, 64%). H NMR (400 MHz, CDCl
3
)
d
(ppm): 8.62 (d,
oxidized product Lyso-NI-SeO and characterized it by H NMR,
C
1
1
1
H, J = 7.2 Hz), 8.44 (d, H, J = 8.0 Hz), 8.39 (d, H, J = 8.0 Hz), 7.39–
NMR, and HRMS. On the other side, we tried to restore the signal of
this oxidized probe with a reducing agent, glutathione (GSH), the
main substance for regulating the redox environment of intercel-
lular compartments. As shown in Fig. 1c, the fluorescence intensity
decreased steadily upon the addition of GSH. This is in line with our
former study that probes based on the redox states of selenide
could be reduced after the reaction with ROS [36,37,42].
1
4
1
7.70(m, 3H), 7.64 (d, H, J = 8.0 Hz), 7.54–7.52 (m, H), 7.46 (d, H,
2
2
4
J = 8.0 Hz), 7.31 (d, H, J = 7.2 Hz), 4.31 (t, H, J = 6.6 Hz), 3.71 (br, H),
.74–2.63 (m, 6H). 1 C NMR (CDCl
63.75, 145.03, 144.48, 136.03, 132.88, 131.61, 131.46, 129.96,
29.82, 128.84, 128.42, 127.66, 126.29, 125.90, 123.42, 123.14,
20.92, 115.20, 111.76, 66.77, 56.20, 53.71, 36.89. HRMS (ESI) Calcd
3
2
1
1
1
3
, 100 MHz) d (ppm): 164.41,
+
for C30
H
28
N
3
O
4
Se [MH ] 574.1245, found 574.1252.
. Results and discussion
.1. The spectroscopic properties of Lyso-NI-Se with HOCl in aqueous
3
.2. Calibration curve for detecting HOCl and the limit of detection
3
In AcOH–AcONa buffer (pH 5.0), Lyso-NI-Se (10
titrated with NaClO and the fluorescence spectra were obtained
with ex = 425 nm (Fig. 2). For the easy and accurate analysis, a
mM) was
3
solutions
l
linear relationship in the calibration curve is crucial. Under the
given conditions, the intensity at 530 nm (F530) was linearly
2 3
In phosphate buffer (50 mM, H O/CH CN = 1/1, V/V, pH 5.0),
Lyso-NI-Se displayed a broad absorption band centered at 452 nm
proportional to the NaClO concentration in the range of 0–10.0 mM
4
À1
À1
3
(
extinction coefficient,
resced. Upon the addition of NaClO, a fluorescence band arose
substantially with a maximum emission wavelength ( em) of
e
= 2.2 Â10 M cm ) and scarcely fluo-
with a regression equation F530/10 = 120.51[NaClO] + 16.68 and a
2
linear coefficient R = 0.9993 (Fig. 2b). Because of the intramolec-
l
ular charge transfer (ICT) nature of naphthalimide fluorophore, the

6
Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
2À
3À
2À
fluorescence of the product was stronger and blue shifted 10 nm in
BuOOH), O , NO, NO , NO ) resulted in no enhancement. Other
ꢁ
this less polarized solvent system (CH
3
CN/H
2
O = 20/1, V/V). The
highly reactive oxygen species (hROS) such as 20 equivalent of OH
À8
À
detection limit of HOCl was calculated to be 1.85 Â10 M from 3
s
/
and peroxynitrite (ONOO ) could introduce some interference
k, where
was the slope of the calibration curve. After the addition of
.2 equiv of NaClO, a 53-fold emission intensity enhancement was
observed, and the fluorescence quantum yield ( ) increased to
.015 (quinine sulphate in 0.1 N H SO as a reference [43]). The
s
was the standard deviation of the blank solutions and k
(2.6- and 4.1-fold of the initial level, respectively). However, the
enhancement was very limited by comparing to the case of 1.2
equivalent of NaClO, in which the signal elevated more than 20-
fold. Importantly, time traces of the signals from parallel samples
maintained unchanged during the testing time (Fig. S1).
1
F
0
2
4
K
f
of Lyso-NI-SeO is really small by comparing to 0.45 of NI-SeO [36],
which is detrimental to detect HOCl with high sensitivity. We
believe that since the nitrogen atom of Lyso-NI-SeO at the 4
positon of 1,8-naphthalimide is conjugated to the adjacent
3
.4. The effect of pH on the fluorescence intensity
To apply this probe in biological systems, the performances of
benzene ring by the “p–p” interaction, the rotation of the aniline
Lyso-NI-Se in acidic or basic solutions were evaluated (Fig. 4). The
fluorescence intensity of Lyso-NI-Se at 540 nm kept unchanged
between pH 2.5–8.5; in the presence of NaClO, fluorescence
augmentation was observed under various pH values. Thus this
probe will function well at lysosomal pH (4.5–5.5) [44,45].
moiety at the excited state may decrease the electron-donating
ability of the nitrogen atom to quench the fluorescence; on the
other hand, the extra methane at the pendant of NI-SeO prevents
this interaction so that NI-SeO exhibits much stronger fluorescence
than Lyso-NI-SeO.
3.3. Selectivity of fluorescent response to HOCl
3.5. Co-staining of Lyso-NI-Se and NR in living cells
The selectivity of Lyso-NI-Se as a fluorescent probe for HOCl was
investigated in phosphate buffer (50 mM, H O/CH CN = 1/1, V/V,
2 3
pH 5.0). As shown in Fig. 3, NaClO triggered a profound
fluorescence signal within a few seconds. This fast responsive
character makes it feasible for real-time detection of HOCl
generated in situ. Exposing this probe to a panel of other
Co-localization experiments were carried out by co-staining
MCF-7 cells (Fig. S2) or RAW264.7 cells (Fig. 5) with Lyso-NI-Se
andneutralred(NR,aconventionaldyeforstaininglysosomes).Since
morpholine has a pKa of 5–6, it can be protonated only in lysosomes
(pH 4.5–5.5) but not in other subcellular structures. Once Lyso-NI-Se
diffuses into lysosomes and is protonated to be more hydrophilic, it
becomes difficult to pass through the membrane of lysosomes [38].
2 2
biologically relevant ROS/RNS (H O ,tert-butylhydroperoxide (t-
Fig. 7. Confocal fluorescent images of endogenous HOCl in RAW264.7 cells (
l
ex = 405 nm,
l
em = 500–550 nm) and the corresponding bright field images. (a) Control, cells were
ꢀ
ꢀ
ꢀ
stained with 5
mM Lyso-NI-Se for 30 min at 37 C, washed 3 times with PBS and incubated for 4.5 h at 37 C. (b) Cells were stained with 5
m
M Lyso-NI-Se for 30 min at 37 C,
ꢀ
washed 3 times with PBS, pretreated with LPS (1
m
g/mL) and IFN-
g
(50 ng/mL) for 4 h and further incubated with PMA (10 nM) for 30 min at 37 C.

Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
7
Lyso-NI-Se was able to permeate cells and palpable discrete
Appendix A. Supplementary data
fluorescence patterns were acquired under a  100 objective lens.
From Fig. 5a and b, it can be clearly observed that the signal of the
green channel excited from the probe coincided with that of the red
channel excited from NR. The intensity profile of the linear regions
of interest(ROI)depicts that signalsfromNRand our probe varied in
close synchrony in both RAW264.7 (Fig. 3e) and MCF-7 cells
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphoto-
chem.2014.10.015.
References
(
Supplementarymaterial).ThePearson’scorrelationcoefficientand
[
1] R.S. Balaban, S. Nemoto, T. Finkel, Mitochondria, oxidants, and aging, Cell 120
2005) 483–495.
2] S.G. Rhee, H , anecessaryevilforcellsignaling, Science312 (2006)1882–1883.
overlapvalueof theco-localizationwere0.85and0.87, respectively;
these high parameters also indicated that Lyso-NI-Se was able to
target lysosomes in living cells.
(
[
2 2
O
[3] C. Kubota, S. Torii, N. Hou, N. Saito, Y. Yoshimoto, et al., Constitutive reactive
oxygen species generation from autophagosome/lysosome in neuronal
oxidative toxicity, J. Biol. Chem. 285 (2010) 667–674.
3.6. Fluorescence imaging of exogenous HOCl in RAW264.7 cells
[4] P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and
disease, Physiol. Rev. 87 (2007) 315–424.
[
5] G. Ferrer-Sueta, R. Radi, Chemical biology of peroxynitrite: kinetics, diffusion,
and radicals, ACS Chem. Biol. 4 (2009) 161–177.
The probe was applied to monitoring exogenous HOCl in the
RAW264.7 cell line. In order to collect the fluorescence changes of
more cells, a  40 objective lens was used to get a larger view. As
shown in Fig. 6, after loading of the probe, the fluorescence image
was almost faint under the present imaging condition; by contrast,
the image of the cells treated with NaClO less than 1 min displayed
stronger fluorescence, again with discrete patterns. In addition,
[6] C. Szabó, H. Ischiropoulos, Radi R. Peroxynitrite, Biochemistry, pathophysiology
and development of therapeutics, Nat. Rev. Drug Discov. 6 (2007) 662–680.
[
[
7] C.C. Winterbourn, M.B. Hampton, J.H. Livesey, A.J. Kettle, Modeling the
reactions of superoxide and myeloperoxidase in the neutrophil phagosome, J.
Biol. Chem. 281 (2006) 39860–39869.
8] D.I. Pattison, M.J. Davies, Absolute rate constants for the reaction of
hypochlorous acid with protein side chains and peptide bonds, Chem. Res.
Toxicol. 14 (2001) 1453–1464.
MTT assay of the RAW264.7 cells incubated with 5
demonstrated that Lyso-NI-Se had low toxicity to cultured cells,
with the survival rate over 95% after 24 h (Fig. S3).
m
M probe
[9] Y.W. Yap, M. Whiteman, N.S. Cheung, Chlorinative stress: an under appreciated
mediator of neurodegeneration? Cell. Signal. 19 (2007) 219–228.
10] Y.W. Yap, M. Whiteman, B.H. Bay, et al., Hypochlorous acid induces apoptosis of
cultured cortical neurons through activation of calpains and rupture of
lysosomes, J. Neurochem. 98 (2006) 1597–1609.
[
[
11] M.J. Steinbeck, L.J. Nesti, P.F. Sharkey, J. Parvizi, Myeloperoxidase and
chlorinated peptides in osteoarthritis: potential biornarkers of the disease, J.
Orthop. Res. 25 (2007) 1128–1135.
3.7. Fluorescence imaging of endogenous HOCl in RAW264.7 cells
[
12] D.I. Pattison, M. Davies, Evidence for rapid inter- and intramolecular chlorine
transfer reactions of histamine and carnosine chloramines: implications for
the prevention of hypochlorous-acid-mediated damage, J. Biochem. 45 (2006)
8152–8162.
13] M.B. Hampton, J.K. Anthony, C.C. Winterbourn, Inside the neutrophil
phagosome: oxidants, myeloperoxidase, and bacterial killing, Blood 92 (1998)
We tried to use the designed probe Lyso-NI-Se to detect
endogenous HOCl production in RAW264.7 cells stimulated by
lipopolysaccharide (LPS)/interferon- (IFN- ) and phorbol 12-
myristate 13-acetate (PMA) [25]. Since the lysosome is
subcellular compartment where MPO and H both exist [46],
g
g
[
a
2 2
O
3
007–3017.
we really wondered that HOCl might also be generated in
lysosomes. However, the fluorescence intensities of cell images
had no significant difference between the control and the
stimulated groups (Fig. 7). One plausible explanation of our
observations is that the detection limit of this probe in lysosomes is
too high to discriminate the elevated level of HOCl produced by
stimulation. Another is that HOCl does not exist in lysosomes,
because this organelle does not generate HOCl and the HOCl flux
from phagosomes might be eliminated by the reducing substances
within the cytoplasm before it reaches lysosomes. In order to study
the mechanism of the lysosome-targetable fluorescent probe for
HOCl, the further theoretical calculations will be needed [47–50].
[14] C.C. Winterbourn, A.J. Kettle, Redox reactions and microbial killing in the
neutrophil phagosome, Antioxid. Redox. Signal. 18 (2013) 642–660.
[
15] C.C. Winterbourn, M.B. Hampton, J.H. Livesey, A.J. Kettle, Modeling the
reactions of superoxide and myeloperoxidase in the neutrophil phagosome:
implications for microbial killing, J. Biol. Chem. 281 (2006) 39860–39869.
16] Y. Zhou, K. Song, R.G. Painter, M. Aiken, J. Reiser, B.A. Stanton, W.M. Nauseef, G.
Wang, Cystic fibrosis transmembrane conductance regulator recruitment to
phagosomes in neutrophils, J. Innate Immun. 5 (2013) 219–230.
[
[17] Y.W. Yap, M. Whiteman, B.H. Bay, Y. Li, F.S. Sheu, R.Z. Qi, C.H. Tan, N.S. Cheung,
Hypochlorous acid induces apoptosis of cultured cortical neurons through
activation of calpains and rupture of lysosomes, J. Neurochem. 98 (2006)
1597–1609.
[18] C.C. Winterbourn, Reconciling the chemistry and biology of reactive oxygen
species, Nat. Chem. Biol. 4 (2008) 278–286.
[19] G. Li, D. Zhu, Q. Liu, L. Xue, H. Jiang, A strategy for highly selective detection and
imaging of hypochlorite using selenoxide elimination, Org. Lett. 15 (2013)
2
002–2005.
[
20] K. Setsukinai, Y. Urano, K. Kakinuma, H.J. Majima, T. Nagano, Development of
novel fluorescence probes that can reliably detect reactive oxygen species and
distinguish specific species, J. Biol. Chem. 278 (2003) 3170–3175.
4. Conclusions
[
21] Y. Koide, Y. Urano, S. Kenmoku, H. Kojima, T. Nagano, Design and synthesis of
fluorescent probes for selective detection of highly reactive oxygen species in
mitochondria of living cells, J. Am. Chem. Soc. 129 (2007) 10324–10325.
In summary, we have designed a lysosome-targetable fluores-
cent probe (Lyso-NI-Se) for HOCl by attaching the morpholine
moiety and the selenide group to a naphthalimide fluorophore.
This probe featured a fast, selective and sensitive fluorescence
response to HOCl and was capable of monitoring exogenous HOCl
in lysosomes of living cells. Attempts of using Lyso-NI-Se to image
HOCl in stimulated RAW264.7 cells failed, probably due to the
absence of endogenous HOCl in lysosomes or the undesirable
detection limit. Future probes for detecting HOCl in lysosomes
with high brightness emission output and sharp changes in terms
of intensity or spectral wavelength are helpful to estimate
endogenous HOCl in lysosomes.
[22] S. Kenmoku, Y. Urano, H. Kojima, T. Nagano, Development of a highly specific
rhodamine-based fluorescence probe for hypochlorous acid and its application
to real-time imaging of phagocytosis, J. Am. Chem. Soc. 129 (2007) 7313–7318.
[
23] R. Zhang, B. Song, Z. Dai, Z. Ye, Y. Xiao, Y. Liu, J. Yuan, Highly sensitive and
selective phosphorescent chemosensors for hypochlorous acid based on
ruthenium(II) complexes, Biosens. Bioelectron. 50 (2013) 1–7.
24] S. Chen, J. Lu, C. Sun, H. Ma, A highly specific ferrocene-based fluorescent probe
for hypochlorous acid and its application to cell imaging, Analyst 135 (2010)
577–582.
[
[25] Z. Sun, F. Liu, Y. Chen, P.K.H. Tam, D. Yang, A highly specific BODIPY-based
fluorescent probe for the detection of hypochlorous acid, Org. Lett. 10 (2008)
2171–2174.
[
26] W. Lin, L. Long, B. Chen, W. Tan, A ratiometric fluorescent probe for hypochlorite
based on a deoximation reaction, Chem. Eur. J. 15 (2009) 2305–2309.
27] P. Panizzi, M. Nahrendorf, M. Wildgruber, P. Waterman, et al., Oxazine
conjugated nanoparticle detects in vivo hypochlorous acid andperoxynitrite
generation, J. Am. Chem. Soc. 131 (2009) 15739–15744.
[
Acknowledgements
[
28] J. Shepherd, S.A. Hilderbrand, P. Waterman, J.W. Heinecke, R. Weissleder, P.
Libby, A fluorescent probe for the detection of myeloperoxidase activity in
atherosclerosis-associated macrophages, Chem. Biol. 14 (2007) 1221–1231.
We are grateful to the National Science Foundation of China (no.
1203192) for financial support.
2

8
Z. Qu et al. / Journal of Photochemistry and Photobiology A: Chemistry 299 (2014) 1–8
[
[
29] Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, Development of an Si– [40] T. Liu, Z. Xu, D.R. Spring, J. Cui, A lysosome-targetable fluorescentprobe for
rhodamine-based far-red to near-infrared fluorescence probe selective for
hypochlorous acid and its applications for biological imaging, J. Am. Chem. Soc.
imaging hydrogen sulphide in living cells, Org. Lett. 15 (2013) 2310.
[41] R.M. Uppu, Synthesis of peroxynitrite using isoamyl nitrite and hydrogen
peroxide in a homogeneous solvent system, Anal. Biochem. 354 (2006)
165–168.
[42] F. Yu, P. Li, G. Li, G. Zhao, T. Chu, K. Han, A near-IR reversible fluorescent probe
modulated by selenium for monitoring peroxynitrite and imaging in living
cells, J. Am. Chem. Soc. 133 (2011) 11030–11033.
133 (2011) 5680–5682.
30] L. Yuan, W. Lin, Y. Xie, B. Chen, J. Song, Fluorescent detection of hypochlorous
acid from turn-on to FRET-based ratiometry by a HOCl-mediated cyclization
reaction, Chem. Eur. J. 18 (2012) 2700–2706.
31] Y. Yang, H.J. Cho, J. Lee, I. Shin, J. Tae, A rhodamine–hydroxamic acid-based
fluorescent probe for hypochlorous acid and its applications to biological
imagings, Org. Lett. 11 (2009) 859–861.
[
[43] J.W. Eastman, Quantitative spectrofluorimetry-the fluorescence quantum
yield of quinine sulphate, Photochem. Photobiol. 6 (1967) 55–72.
+
[
[
[
32] J. Zhang, X. Wang, X. Yang, Colorimetric determination of hypochlorite with
unmodified gold nanoparticles through the oxidation of a stabilizer thiol
compound, Analyst 137 (2012) 2806–2812.
[44] T.J. Rink, R.Y. Tsien, T. Pozzan, Cytoplasmic pH and free magnesium(2 ) in
lymphocytes, J. Cell Biol. 95 (1982) 189–196.
[45] A.P. de Silva, H.Q.N. Gunaratne, P.L.M. Lynch, A.J. Patty, G.L. Spence,
Luminescence and charge transfer. Part 3. The use of chromophores with ICT
(internal charge transfer) excited states in the construction of fluorescent PET
(photoinduced electron transfer) pH sensors and related absorption pH
sensors with aminoalkyl side chains, J. Chem. Soc. Perkin Trans. 2 (1993)
1611–1616.
33] W. Wu, Z. Li, L. Yang, J. Han, S. Han, A self-referenced nanodosimeter for
reaction based ratiometric imaging of hypochlorous acid in living cells, Chem.
Sci. 4 (2013) 460–467.
34] T. Guo, L. Cui, J. Shen, R. Wang, W. Zhu, Y. Xu, X. Qian, A dual-emission and large
stokes shift fluorescence probe for real-time discrimination of ROS/RNS and
imaging in live cells, Chem. Commun. 49 (2013) 1862–1864.
35] S. Liu, S. Wu, Hypochlorous acid turn-on fluorescent probe based on oxidation
of diphenyl selenide, Org. Lett. 15 (2013) 878–881.
[46] J. Jing, J.L. Zhang, Combining myeloperoxidase (MPO) with fluorogenic ZnSalen
to detect lysosomal hydrogen peroxide in live cells, Chem. Sci. 4 (2013)
2947–2952.
[
[
36] Z. Lou, P. Li, Q. Pan, K. Han, A reversible fluorescent probe for detecting
hypochloric acid in living cells and animals: utilizing a novel strategy for
effectively modulating the fluorescence of selenide and selenoxide, Chem.
Commun. 49 (2013) 2445–2447.
[47] G.J. Zhao, K.L. Han, Hydrogen bonding in the electronic excited state, Acc.
Chem. Res. 45 (2012) 404–413.
[48] G.J. Zhao, J.Y. Liu, L.C. Zhou, K.L. Han, Site-selective photoinduced electron
transfer from alcoholic solvents to the chromophore facilitated by hydrogen
bonding: a new fluorescence quenching mechanism, J. Phys. Chem. B 111
(2007) 8940–8945.
[49] G.Y. Li, G.J. Zhao, Y.H. Liu, K.L. Han, G.Z. He, TD-DFT study on the sensing
mechanism of a fluorescent chemosensor for fluoride: excited-state proton
transfer, J. Comput. Chem. 31 (2010) 1759–1765.
[
37] B. Wang, P. Li, F. Yu, P. Song, X. Sun, et al., A reversible fluorescence probe based
on Se–BODIPY for the redox cycle between HClO oxidative stress and H2S
repair in living cells, Chem. Commun. 49 (2013) 1014–1016.
[
38] L. Wang, Y. Xiao, W. Tian, L. Deng, Activatable rotor for quantifying lysosomal
viscosity in living cells, J. Am. Chem. Soc. 135 (2013) 2903–2906.
39] H. Yu, Y. Xiao, L. Jin, A lysosome-targetable and two-photon fluorescent probe
for monitoring endogenous and exogenous nitric oxide in living cells, J. Am.
Chem. Soc. 134 (2012) 17486–17489.
[
[50] G.Y. Li, Y.H. Li, H. Zhang, G.H. Cui, Time-dependent density functional theory
study on a fluorescent chemosensor based on CHÁ Á Á Hydrogen-bond
interaction, Commun. Comput. Chem. 1 (2013) 88–98.