A 1,8-naphthalimide-derived turn-on fluorescent probe for imaging lysosomal nitric oxide in living cells

Article abstract of DOI:10.1016/j.cclet.2016.06.016

Nitric oxide has played an important role in many physiological and pathological processes as a kind of important gas signal molecules. In this work, a new fluorescent probe LysoNO-Naph for detecting NO in lysosomes based on 1,8-naphthalimide was reported. LysoNO-Naph has sub-groups of o-phenylenediamine as a NO reaction site and 4-(2-aminoethyl)-morpholine as a lysosome-targetable group. This probe exhibited good selectivity and high sensitivity (4.57?μmol/L) toward NO in a wide pH range from 4 to 12. Furthermore, LysoNO-Naph can be used for imaging NO in lysosomes in living cells.

Full text of DOI:10.1016/j.cclet.2016.06.016

Chinese Chemical Letters 27 (2016) 1554–1558
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
A 1,8-naphthalimide-derived turn-on fluorescent probe for imaging
lysosomal nitric oxide in living cells
Wei Feng a,b, Qing-Long Qiao b,c, Shuang Leng b, Lu Miao b, Wen-Ting Yin b, Li-Qiu Wang a,
*
,
Zhao-Chao Xu b,
*
a College of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China
b State Key Laboratory of Fine Chemicals, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
c State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Nitric oxide has played an important role in many physiological and pathological processes as a kind of
important gas signal molecules. In this work, a new fluorescent probe LysoNO-Naph for detecting NO in
lysosomes based on 1,8-naphthalimide was reported. LysoNO-Naph has sub-groups of o-phenylene-
diamine as a NO reaction site and 4-(2-aminoethyl)-morpholine as a lysosome-targetable group. This
Received 22 April 2016
Received in revised form 10 May 2016
Accepted 16 May 2016
Available online 17 June 2016
probe exhibited good selectivity and high sensitivity (4.57
mmol/L) toward NO in a wide pH range from
4 to 12. Furthermore, LysoNO-Naph can be used for imaging NO in lysosomes in living cells.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Fluorescent probe
Nitric oxide
Lysosome localized
Imaging in living cells
1. Introduction
Nitric oxide (NO) as a uncharged free radical exists everywhere
in space [16,17]. Because of its highly chemical reactivity, early
Fluorescence imaging has unique advantages to study cellular
functional molecules in vitro and/or in vivo [1–3]. By attaching a
sub-cellular organelle specific group, fluorescent probes can be
used to detect the localization and monitor the movement of target
biomolecules [4–7]. Lysosomes are round-shaped organelles that
contain acid hydrolase enzymes that break down waste materials
and cellular debris. The interior of the lysosomes is acidic at pH
4.8 compared to the slightly basic cytosol at pH 7.2. Lysosomes
were traditionally treated only as the cell recycling centers. They
can make the senescent cellular debris and needless macromo-
lecules into small molecules, and then the cells utilize the small
molecules for living [8]. In recent years, lysosomes have been
recognized as an more important organelle in cells. They are
related to many diseases such as cancer, neurodegenerative
disorders, and cardiovascular diseases. It is extremely urgent to
make real-time detection and imagining of lysosomes as well as
inside molecules. In recent years, fluorescent probes for staining
lysosomes [9–11] or image lysosomal H2S [12], NO, [13] HOCl [14]
and pH [15] have been reported.
studies recognized NO as a kind of poison without other biological
significance. As time goes on, we started to realize that NO is an
indispensable messenger molecule in the cardiac disorders,
immune system disorder and neurodegeneration [18–20]. It makes
white blood cells kill tumor cells and bacteria, and much like
neurotransmitter, it can dilate blood vessels. Disclosing the
distribution of NO and its relationship with bodily agents will
be critical for the treatment for the disorders mentioned above.
Some fluorescent probes, including o-phenylenediamine based
fluorescent probes [21–29] and copper complex based compounds
[30], have been reported to detect NO with some successful
applications to image NO in living cells.
In this paper, we reported a new fluorescent probe LysoNO-
Naph to detect NO in lysosomes. Derived from 4-bromo-1,8-
naphthalic anhydride, the NO reactive site o-phenylenediamine
and a lysosome target group 4-(2-aminoethyl)-morpholine were
introduced to the fluorophore. This probe can be easily obtained
with high yield. As shown in Scheme 1, LysoNO-Naph reacted with
NO to form a triazole ring and simultaneously displayed a turn-on
blue fluorescence. Its propreties of imaging lysosomal NO were
also studied. The experimental details are given in Supporting
information.
*
Corresponding authors.
E-mail addresses: liqiuwang@tom.com (L.-Q. Wang), zcxu@dicp.ac.cn (Z.-C. Xu).
http://dx.doi.org/10.1016/j.cclet.2016.06.016
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

W. Feng et al. / Chinese Chemical Letters 27 (2016) 1554–1558
1555
Scheme 1. Synthesis of LysoNO-Naph and the reaction process with NO.
2. Experimental
2.3. Synthesis of LysoNO-Naph
2.1. Materials and instruments
To a solution of 3,4-diamino-1,8-naphthalic anhydride (200 mg,
0.88 mmol) in 20 mL 2-methoxyethanol was added 2.0 eq 4-(2-
Unless otherwise noted, all reagents were obtained from
Aldrich and used without further purification. 1H NMR and 13C
NMR spectra were recorded on a Burker 400 MHz spectrometer.
UV-visible absorption spectra were collected on Agilent Cary
60 UV/VIS spectrophotometer. Fluorescence emission spectra were
performed on Cary Eclipse Fluorescence Spectrophotometer (Serial
No. FL0812-M018). Compounds 2–4 were synthesized with a
modified method according to [31].
aminoethyl)-morpholine (231 mL, 1.76 mmol). The mixture was
then heated at 125 8C for 5 h and monitored by TLC. After the
completion of the reaction, the residue was purified by silica gel
column (CH2Cl2:CH3OH = 20:1) to give 150 mg LysoNO-Naph as
red powder in 51% yield. M. p. 225–227 8C. 1H NMR (400 MHz,
DMSO-d6): d 8.49 (d, 1H, J = 8.3 Hz), 8.20 (d, 1H, J = 7.0 Hz), 7.92 (s,
1H), 7.58–7.52 (t, 1H), 6.51 (s, 2H), 5.15 (s, 2H), 4.14 (t, 2H,
J = 6.9 Hz), 3.56–3.51 (m, 4H), 2.53–2.50 (m, 2H), 2.45 (s, 4H). 13C
HPLC-MS analysis was performed on Agilent 6540 UHD
Accurate-Mass Q-TOF LC/MS using an HPLC system composed of
a pump (Agilent ZORBAX Eclipse Plus C18 2.1 mm  50 mm) and a
LysoNO-Naph detector (254 nm).
NMR (100 MHz, DMSO-d6): d 164.50 (s), 163.57 (s), 136.88 (s),
131.00 (s), 128.47 (s), 127.70 (s), 124.19 (s), 123.75 (s), 122.11 (s),
121.31 (s), 120.06 (s), 108.76 (s), 66.68 (s), 56.32 (s), 53.89 (s),
36.82 (s). HRMS (ESI) calcd. for C18H21N4O3 [M + H+] 341.1614,
found 341.1610.
2.2. Synthesis of compound 2–4
2.4. Culture of CHO cells and fluorescent imaging:
2.2.1. Synthesis of compound 2
NaNO3 (2.0 g, 23.53 mmol) was carefully added to a suspension
of 4-bromo-1,8-naphthalic anhydride (5.0 g, 18.1 mmol) in con-
centrated sulphuric acid (30 mL). The solution was stirred for 3 h at
0 8C, and then moved to room temperature for 1 h. The solution
was poured into 300 mL of ice-water, filtered to give yellow solid.
The crude product was recrystallized in glacial acetic acid to yield
product as yellow solid (3.76 g, 65%). 1H NMR (400 MHz, DMSO-
CHO cells was hatched in an atmosphere of 5% CO2 and 95% air
in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) at
37 8C. The cells were seeded in 24-well flat-bottomed plates and
then incubated for 72 h at 37 8C under 5% CO2. After adding
LysoNO-Naph (5
30 min. Then use phosphate-buffered saline (PBS, 10 mmol/L) to
wash cells for three times. Added 100 mol/L of SNP (release NO,
50 mol/L) and Neutral Red (2 mol/L) to the cells, incubate them
mmol/L) to the cells, incubate them for another
m
d6):
d
8.93 (s, 1H), 8.84 (d, 1H, J = 8.6 Hz), 8.75 (d, 1H, J = 7.9 Hz),
m
m
8.23–8.16 (m, 1H). MS (EI) calcd. for C12H4BrNO5 [M+] 320.93,
found 320.93.
for 30 min. Fluorescence imaging was observed under a confocal
microscopy (Olympus FV1000) with a 60Â objective lens.
2.2.2. Synthesis of compound 3
3. Results and discussion
After a mixture of compound 2 (2 g, 6.32 mmol) and DMF
(25 mL) was stirred at room temperature for 10 minutes, NaN3
(0.608 g, 9.35 mmol) was carefully added, and the mixture was
stirred for 3 h at room temperature. The solution was poured into
200 mL of ice-water, and the precipitate was filtered and washed
with water. After drying, compound 3 was obtained (1.2 g, 68%). 1H
As lysosomes have an acid interior with pH from 4.0 to 6.0,
lysosome-targetable fluorescent probes must remain unaffected
fluorescence without the interaction with analyte at least in this
pH range. So, the pH effect on the absorption and fluorescence
of LysoNO-Naph were firstly studied. As shown in Fig. 1, the
maximum absorption peak of LysoNO-Naph at 462 nm remained
stable in the pH range from pH 3.80 to 12.54. Accordingly, LysoNO-
Naph did not fluoresce in this pH range. In much more acidic
solutions, the maximal absorption peak moved to shorter
wavelength. And in much more basic solutions, the maximal
absorption peak reminded but with decreased fluorescence
intensity. Hence, LysoNO-Naph is suitable to be applied to monitor
lysosomal NO, since it is stable in pH from 4.0 to 6.0 without
any changes. The polarity effects on absorption and fluorescence
were also examined. As indicated in Figs. S2 and S3 (see Supporting
information), LysoNO-Naph is a typical intramolecular charge-
transfer (ICT) type of fluorescent dyes. With the increase of
polarity, the absorption and emission wavelengths would be red-
shifted.
NMR (400 MHz, DMSO-d6):
J = 7.2 Hz), 8.08 (s, 1H), 7.69 (t, 1H, J = 7.9 Hz). MS (EI) calcd. for
12H4N4O5 [M+] 284.02, found 284.02.
d 8.69 (d, 1H, J = 8.5 Hz), 8.34 (d, 1H,
C
2.2.3. Synthesis of compound 4
After a mixture of SnCl2 (3 g, 13.31 mmol) and concentrated
hydrochloric acid (8 mL) was stirred at room temperature for
10 min, compound 3 was slowly added, and the mixture was
stirred for 40 minutes at 50 8C. Then stirred at 80 8C with ethanol
(8 mL) for another 3 h. After the reaction, mixture was cooled
down to room temperature, the precipitate was filtered and
washed with water. After drying, red compound 4 was obtained
(0.54 g, 81%).1H NMR (400 MHz, DMSO-d6):
d 8.58 (d, 1H,
J = 8.5 Hz), 8.21 (d, 1H, J = 7.1 Hz), 7.89 (s, 1H), 7.62–7.56 (m,
1H), 6.89 (s, 2H), 5.28 (s, 2H). MS (ESI) calcd. for C12H9N2O3
[M + H+] 238.13, found 238.13.
The absorption and fluorescence spectra of LysoNO-Naph with
the addition of NO were obtained in mixture solutions of

1556
W. Feng et al. / Chinese Chemical Letters 27 (2016) 1554–1558
40
35
30
25
20
15
10
5
y = -6.7 + 48.8 x
2
0.06
0.04
0.02
R
= 0.99472
A462
0.10
pH = 0.76-3.8
600
0
5
10
pH = 3.8-12.54
pH = 13.03
pH = 13.24
pH
0.3
0.4
0.5
0.6
0.7
0.8
0.9
[SNP]/[Probe]
A
0.05
400
200
0
0.00
300
400
Wavelength (nm)
Fig. 1. Effects of pH on the absorption of LysoNO-Naph.
500
400
500
Wavelength (nm)
acetonitrile and PBS buffer s (1:1, v/v, pH 7.4, 20 mmol/L). LysoNO-
Naph has a main absorption band at 462 nm and a second
absorption band at 368 nm (Fig. 2a). When 25 equiv. of SNP (NO
donor) were added to the solution of LysoNO-Naph, a significant
decrease in the 462 nm absorption and a blue-shifted absorption
band centered at 368 nm, which was attributed to the formation
of compound LysoNO-Naph-1 and increased in intensity, were
observed with an isoabsorption point at 405 nm. In the same time,
the fluorescence emission band centered at 460 nm appeared
Fig. 3. (a) Fluorescence changes and (b) fluorescence ratio (F454/F0) changes of
LysoNO-Naph (10 mol/L) upon addition of SNP (0–1 eqiv.).
m
including NO, NO2À, NO3À, H2O2 and ClOÀ. As shown in Fig. 5, when
LysoNO-Naph was added with 25 equiv. SNP, a great fluorescent
increase at 454 nm and a great absorption band at 368 nm were
observed. With the addition of 100 equiv. of NO2À, NO3À, H2O2 and
ClOÀ, no obvious changes in fluorescence and absorption were
observed. In this regard, LysoNO-Naph has a high selectivity for
NO.
We next applied LysoNO-Naph for fluorescence imaging of NO
in CHO cells (Chinese Hamster Ovary). After incubation with
mmol/L LysoNO-Naph in culture medium for 30 min, cells were
and increased in intensity (FF = 0.23) (Fig. 2b). We found that if
the concentration of added SNP was 25 equiv., the reaction can
be finished within 20 min (insets in Fig. 2). With much more SNP
like 50 equiv. (Fig. S1 in Supporting information), the reaction can
be finished in a shorter time (10 min). Then we decided to use
25 equiv. of SNP to examine the performance of LysoNO-Naph in
following experiments.
Fig. 3 displayed the titration experiments of LysoNO-Naph with
the addition of 0–1 eqiv. SNP. There was a good linearity between
the ratio in the fluorescent intensity (F454/F0) and concentrations
of SNP (in the range of 0–1 eqiv.) with a detection limit to
5
washed with phosphate buffered saline (PBS, 10 mmol/L, pH 7.4)
for three times to remove needless probe. There was a very weak
fluorescence existed in confocal image (Fig. 6a). Then 50
mmol/L
SNP was added and treated for another 30 min, and a strong blue
fluorescence was observed, indicating the detection of cellular NO
by LysoNO-Naph (Fig. 6b). In order to identify organelles from the
fluorescence imaging of LysoNO-Naph in cells, we then carried out
fluorescence localization by co-staining cells with commercially
available organelle-specific dyes (Fig. 6c and d). The highly blue
fluorescent regions with LysoNO-Naph (Fig. 6b) and signals from
NR (Fig. 6c) overlapped perfectly (Fig. 6d), indicating that these
4.57
mmol/L (Fig. 3). Moreover, the light blue fluorescence change
can be easily recognized by naked eyes. The reaction product was
analyzed by HPLC-MS. As shown in Fig. 4, the blue fluorescence
product was proved to be LysoNO-Naph-1.
We also tested the selectivity of LysoNO-Naph with different
kind of reactive species (ROS) and reactive nitrogen species (RNS)
(b)
(a)
20
15
10
5
600
A368
A462
FI460
400
200
600
0
0.10
0.05
0.00
0
0
5
10 15 20 25 30
0
5
10 15 20 25 30
Time (min)
Time (min)
400
200
0
A
400
450
500
550
600
300
400
Wavelength (nm)
500
Wavelength (nm)
mmol/L) with SNP (25 eqiv.) from 0 to 30 min.
Fig. 2. (a) Absorption and (b) fluorescence of LysoNO-Naph (10

W. Feng et al. / Chinese Chemical Letters 27 (2016) 1554–1558
1557
Fig. 4. HPLC-MS of the reaction product of LysoNO-Naph (10
m
mol/L) and SNP (25 eqiv.).
mol/L) with the addition of ClO-, H2O2, NO3À, NO2 (100 eqiv.) and SNP(25 eqiv.).
À
Fig. 5. (a) Absorption and (b) fluorescence of LysoNO-Naph (10
m
Fig. 6. Fluorescence images of CHO cells incubated with 10
mmol/L LysoNO-Naph and NO. Cells treated with LysoNO-Naph (a) in the absence and (b) presence of 50 eqiv. SNP.
(c) Image of lysosome tracker (Neutral Red). (d) Fluorescence co-localization imaging of LysoNO-Naph and Neutral Red. (e) Bright field image.

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In conclusion, we have reported a fluorescent probe LysoNO-
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The probe can be obtained easily with a high yield. Its insensitivity
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Acknowledgment
We thank financial supports from the National Natural Science
Foundation of China (Nos. 21276251, 21506206, 21402191,
21502189), the 100 talents program funded by Chinese Academy
of Sciences, Dalian Cultivation Fund for Distinguished Young
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