A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis, photophysical properties and cell imaging

Article abstract of DOI:10.1016/j.cclet.2014.05.020

PAHPN, a naphthalimide-based mitotracker with reasonable two-photon excitation emission activity and polarity-sensitive fluorescence properties has been efficiently synthesized and studied in two-photon, co-localization, and FLIM imaging.

Full text of DOI:10.1016/j.cclet.2014.05.020

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CCLET 3001 1–5
Chinese Chemical Letters xxx (2014) xxx–xxx
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
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Original article
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4
A two-photon mitotracker based on a naphthalimide fluorophore:
Synthesis, photophysical properties and cell imaging
c
c,
Q1 Yong Dai a,c, Bao-Kuo Lv b, , Xin-Fu Zhang , Yi Xiao
* *
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a Department of Criminal Science and Technology, Sichuan Police College, Luzhou 646000, China
b Liaoning Water Environment Monitoring Center, Shenyang Branch, Shenyang 110005, China
c State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
A R T I C L E I N F O
A B S T R A C T
Article history:
PAHPN, a naphthalimide-based mitotracker with reasonable two-photon excitation emission activity
and polarity-sensitive fluorescence properties has been efficiently synthesized and studied in two-
photon, co-localization, and FLIM imaging.
Received 27 March 2014
Received in revised form 26 April 2014
Accepted 29 April 2014
Available online xxx
ß 2014 Bao-Kuo Lv. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords:
Mitotracker
Mitochondria
Namphthalimide
Two-photon
Fluorescence imaging
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1. Introduction
emerging imaging technique using pulsed NIR excitation can be 30
a superior alternative to confocal microscopy (one photon 31
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Mitochondria, as cell’s power producers, are important
organelles for cellular respiration that ultimately generates fuel
for the cellular activities [1]. In addition, mitochondria are involved
in other tasks such as signaling, cellular differentiation, cell death,
as well as the control of the cell cycle and cell growth [2]. In
particular, mitochondria’s key role in activating apoptosis has
attracted much attention [3]. Mitochondria are highly dynamic
organelles that continuously move, divide and fuse in a highly
regulated fashion during various cellular processes [4]. For
example, mitochondrial fission accompanies apoptotic cell death
and appears to be important for the progression of the apoptotic
pathways [5].
Mitotrackers [6], namely mitochondria targeted fluorescent
probes, have proved valuable tools to visualize mitochondria’s
dynamic changes during apoptosis and other cellular processes.
However, the common mitotrackers, e.g., Rhodamine 123 and
tetramethylrhodamine methyl ester (TMRM), are not efficient
two-photon fluorophores, which restricted their application in
TPEE (two photon excitation emission) microscopy. This newly
imaging) due to its deeper tissue penetration (>500 mm), 32
efficient light detection, and reduced phototoxicity [7,8]. Due 33
to its imaging mechanism, it also provides advantages such as 34
highly localized excitation and prolonged observation time. 35
These are desired imaging properties for trackers and the 36
development of two-photon mitotrackers represents a critical 37
priority.
38
Herein, we have developed PAHPN, a new mitotracker with 39
reasonable TPEE activity. There are two key aspects in our design. 40
Firstly, we chose 4-pyrrolidino-1,8-naphthalimide as the fluor- 41
ophore because its TPEF activity is foreseeable since another 4- 42
amino naphthalimide probe showing strong TPEE has been 43
reported by ourselves very recently [9]. Secondly, a triphenylpho- 44
sphonium (TPP) moiety has been attached to the naphthalimide 45
fluorophore via a flexible long alkyl chain, which generates a 46
positively charged but highly lipophilic molecule that tends to 47
accumulate in mitochondrial inner membranes at negative 48
potentials [10,11].
49
As shown in Scheme 1, the synthesis of PAHPN can be achieved 50
from 4-Br-1,8-naphthalic anhydride in four steps, namely, 51
nucleophilic substitution, condensation, acylation and quarter- 52
nization. Each reaction is facile under mild conditions, thanks to 53
Qian’s pioneering work on the modifications of the naphthalimide 54
*
Corresponding authors.
E-mail addresses: lvbaokuo1987@163.com (B.-K. Lv),
platform [12].
55
xiaoyi@dlut.edu.cn (Y. Xiao).
http://dx.doi.org/10.1016/j.cclet.2014.05.020
1001-8417/ß 2014 Bao-Kuo Lv. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: Y. Dai, et al., A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis,
photophysical properties and cell imaging, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.020

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Y. Dai et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Scheme 1. Synthesis of PAHPN. (a) Pyrrolidine, 2-methoxyethanol, reflux, 72%. (b) Hexamethylenediamine, ethanol, reflux, 80%. (c) Bromoacetic acid, DCC, dichloromethane,
r.t., 94%. (d) Triphenylphosphine, dichloromethane, r.t., 35%.
56
2. Experimental
(d, 1H, J = 8.8 Hz), 7.54 (t, 1H), 8.42 (d, 1H), 8.58 (t, 2H). 13C NMR
(100 MHz, CDCl3): 25.1, 25.7, 26.3, 28, 29.1, 29.5, 34, 39.9, 40.2,
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d
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Culture of cells and fluorescent imaging: MCF7 (human breast
carcinoma) cells, RAW 264.7 (macrophages cells) and COS-7 cells
were obtained from Institute of Basic Medical Sciences (IBMS) of
Chinese Academy of Medical Sciences (CAMS). All cell lines were
maintained under standard culture conditions (atmosphere of 5%
CO2 and 95% air at 37 8C) in RPMI 1640 medium, supplemented
with 10% FBS (fetal calf serum).
Cells were grown in the exponential phase of growth on 35 mm
glass-bottom culture dishes (Ø20 mm) for 1–2 days to reach 70–
90% confluency. These cells were used in co-localization experi-
mentation. The cells were washed three times with RPMI 1640, and
then incubated with 2 mL of RPMI 1640 containing probes
49.4, 53.3, 108.3, 110.6, 122.7, 123.2, 131.2, 132.1, 133.2, 152.6,
164.3, 165, 165. HRMS (MALDI-TOF) (m/z): Calcd. for
C
24H28BrN3O2: 485.1314, found: 486.1394 ([M+H+]+).
PAHPN: a mixture of compound 3 (0.12 g, 0.247 mmol) and
triphenylphosphine was stirred in CH2Cl2 at room temperature for
5 h. After the CH2Cl2 was removed under reduced pressure, the
residue was purified by silica gel column chromatography using
eluent CH2Cl2/MeOH (20/1, v/v). A yellow solid was obtained
(0.065 g, 35.2%). Mp 94–96 8C. 1H NMR (400 MHz, CDCl3):
d 1.31–
1.16 (m, 6H), 1.62 (m, 2H), 2.09 (s, 4H), 3.05 (d, 2H, J = 6 Hz), 3.76 (s,
4H), 4.09 (t, 2H), 5.02 (d, 2H, J = 14 Hz), 6.79 (d, 1H, J = 8.8 Hz), 7.50
(t, 1H), 7.64 (m, 6H), 7.82 (m, 9H), 8.38 (d, 1H, J = 8.8 Hz), 8.55 (m,
2H), 9.29 (s, 1H). 13C NMR (100 MHz, CDCl3):
d 26.1, 26.8, 26.9,
28.1, 29, 31.9, 32.5, 40.1, 40.2, 53.2, 108.6, 110.7, 118.1, 119, 122.5,
123.1, 130.1, 130.2, 131.0, 133.4, 134.1, 134.3, 135.0, 152.7, 162.1,
164, 164.8. HRMS (MALDI-TOF) (m/z): Calcd. for C42H43BrN3O3P:
747.2225, found: 668.2996 ([MÀBrÀ]+).
(1
m
mol/L) in an atmosphere of 5% CO2 and 95% air for 3 min at
37 8C. Cells were washed twice with 1 mL of PBS at room
temperature and observed under a confocal microscopy (Olympus
FV1000).
The 400 MHz 1H NMR and 100 MHz 13C NMR spectra were
collected at room temperature and were given in supporting
information. Melting points were obtained with a capillary melting
point apparatus in open-ended capillaries and were uncorrected.
Chromatographic purifications were conducted using silica gel. All
solvent mixtures are given as volume/volume ratios.
Compound 1: 4-Bromo-1,8-naphthalic anhydride (10 g,
36.2 mmol) was dissolved in ethylene glycol monomethyl ether
under reflux. Pyrrolidine (5 mL, 66.4 mmol) was added in four
portions in 2 h. After the addition of pyrrolidine, the mixture was
refluxed for one more hour. After the reaction mixture was cooled
to room temperature, the yellow solid was collected (7 g, 72.4%).
Compound 2: Compound 1 (1 g, 3.74 mmol) and hexamethy-
lenediamine (1.3 g, 11.23 mmol) were refluxed in 10 mL ethanol.
The reaction mixture was cooled to room temperature. After the
ethanol was removed under reduced pressure, the residue was
recrystallized from ethanol to give the compound 2 (1 g, 80%). Mp.
3. Results and discussion
123
PAHPN exhibits polarity-sensitive fluorescence properties. Its
absorption and emission spectra in various solvents are shown in
Fig. 1, and the basic data are listed in Table 1. Briefly, with the
increase in polarity, the fluorescence spectra red-shift to longer
wavelength range and the fluorescence quantum yields decrease
sharply. For example, in toluene, the emission maximum is at
500 nm and the quantum yield is 0.96, while in acetonitrile, the
emission peak moves to 541 nm and the quantum yield declines to
0.14. However, the absorption properties are less dependent on
solvent polarity. Although the absorption spectra also shift in
various solvents, the difference in molar extinction coefficients is
not as significant as that of fluorescence quantum yields. The
sharply different fluorescence is an advantage for PAHPN’s
application in mitochondria imaging since those PAHPN molecules
localized in mitochondrial inner membrane would emit strong
fluorescence due to the nonpolar lipophilic environment while the
background noise from some PAHPN distributed in other aqueous
intracellular compartments would be low. Its fluorescence life
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60–61.4 8C. 1H NMR (400 MHz, CDCl3):
2.1 (d, 6H), 2.71 (t, 2H), 3.77 (s, 4H), 4.16 (t, 2H), 6.80 (d, 1H), 7.52 (t,
1H), 8.41 (d, 1H), 8.58–8.54 (m, 2H). 13C NMR (100 MHz, CDCl3):
26.2, 26.7, 27, 28.2, 29.8, 33.2, 40.1, 42.1, 53.3, 108.7, 111, 122.8,
123.2, 131.1, 131.3, 132, 133.5, 152.8, 164.2, 165.0. HRMS (MALDI-
TOF) (m/z): Calcd. for C22H27N3O2: 365.2103, found: 366.2192
([M+H+]+).
d 1.48 (m, 6H), 1.74 (s, 2H),
d
times (t) in various solvents are given in Fig. 1, and the average t
values are listed in Table 1. Fluorescence life time shows a decrease
with the increase in polarity, which can be used to measure the
polarity of mitochondria.
The sensitivity of fluorescence toward polarity could be
explained by TICT (twisted intramolecular charge transfer, or
twisted ICT) mechanism [13]. PAHPN’s fluorophore, 4-pyrrolidino-
1,8-naphthalimide accords with the standard TICT structure: the
strong electron donor (pyrrolidino) is connected to the strong
Compound 3: Compound 2 (0.2 g, 0.55 mmol), bromoacetic acid
(47.24
mL, 0.66 mmol) and DDC (160 mg, 0.66 mmol) were stirred
in CH2Cl2 at room temperature for 6 h. The insoluble materials
were filtered off and the filtrate was evaporated to provide the
compound 3 (0.25 g, 94.0%). Mp 79–80.2 8C. 1H NMR (400 MHz,
CDCl3):
d 1.15 (m, 2H), 1.74 (m, 4H), 1.93 (d, 2H), 2.11 (s, 4H), 3.29
(m, 2H), 3.79 (s, 4H), 3.9 (s, 2H), 4.18 (t, 2H), 6.69 (s, 1H), 6.82
Please cite this article in press as: Y. Dai, et al., A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis,
photophysical properties and cell imaging, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.020

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Y. Dai et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
3
a
b
0.2
0.1
0.0
toluene
THF
ethyl acetate
acetone
acetonitrile
toluene
THF
300
ethyl acetate
acetone
acetonitrile
200
100
0
450
500
550
600
650
350
400
450
500
Wavelength/nm
Wavelength/nm
1.0
0.8
0.6
0.4
0.2
0.0
c
d
8
toluene
THF
ethyl acetate
acetone
6
4
2
acetonitrile
30
35
40
45
30
40
50
60
-1
Time/ns
ET/Kcal*mol
Fig. 1. Absorption (a) and emission (b) spectra of PAHPN in solvents of different polarities; fluorescence life time of PAHPN in solvents of different polarities (c) and (d).
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electron acceptor (naphthalimide) by
a
single bond. Upon
The applicability of PAHPN as a two-photon fluorescent cell 171
marker has been confirmed by two-photon microscopy imaging 172
experiments. As shown in Fig. 3, strong TPEE has been observed 173
in MCF-7 cells stained with PAHPN. The distribution of 174
fluorescence clearly indicates that the PAHPN molecules are 175
not uniformly dispersed but localized in certain areas. Mito- 176
chondria usually assemble into networks, although single ones 177
have an ellipsoid shape [6]. In Fig. 3, fluorescent networks are 178
easily visualized with high resolution because the areas 179
surrounding these networks are almost ‘dark’ (without back- 180
ground noise). Thus, the quality of two-photon imaging is high. 181
excitation of such a fluorophore, the single bond will be twisted
quickly so that the donor plane and the acceptor plane become
perpendicular to each other. Such a twisted configuration in
excited state means a charge separation between the donor and
acceptor, or a complete charge transfer. This movement of the
single bond consumes the excitation energy and thus quenches the
fluorescence sharply. As we know, highly polar solvents can
stabilize and thus promote TICT state, while nonpolar solvents
destabilize and disfavor TICT.
PAHPN demonstrates
a reasonable two-photon excitation
activity. As shown in Fig. 2, the two-photon excitation spectrum
of PAHPN in toluene exhibits a broad band from 700 to 1000 nm
and the maximum is at 820 nm, which is approximately the double
of the one-photon absorption maximum. The TPEE activity across
90
60
30
0
section (
F
Â
d, the product of F and d) at 820 nm is around 90 GM,
which is a reasonable value and higher than that of several
common fluorophores. In other word, the intrinsic brightness of
PAHPN’s TPEE is sufficient for two-photon microscopic applica-
tions.
Table 1
Spectral data of PAHPN.
Solvent
lab (nm)
lem (nm)
Fa
e
t (ns)
Toluene
THF
436
436
435
443
446
500
505
506
517
523
0.96
0.98
0.97
0.20
0.14
15,400
15,900
15,900
17,700
17,400
7.19
5.71
5.13
1.87
1.67
Ethyl acetate
Acetone
CH3CN
700
800
Wavelength/nm
900
1000
a
Quinine sulfate (
F
= 0.55) as reference.
Fig. 2. Two-photon excitation spectrum of PAHPN in toluene.
Please cite this article in press as: Y. Dai, et al., A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis,
photophysical properties and cell imaging, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.020

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Y. Dai et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Fig. 3. Two photon fluorescence image of MCF-7 cells incubated with PAHPN (1
mmol/L) for 3 min at 37 8C. (a) Fluorescence image (lex = 840 nm, lem = 520–560 nm). (b)
Overlay of fluorescence image and brightfield image.
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Actually, we have also applied PAHPN in one-photon confocal
imaging (Fig S1 in supporting information) and have found that
there is no difference in imaging quality between two-photon
and one-photon microcopies.
PAHPN and TMRM (or mitotracker deep red) are localized in
similar intracellular areas, namely, mitochondria. Similar
colocalization results are also obtained in RAW264.7 and
COS-7 cells, as shown in the supporting information (Figs. S2
and S3) In other words, PAHPN shows stable localization in
mitochondria of different cell lines. Thus, it can be concluded
that PAHPN is mitochondria-targeted.
Finally, PAHPN is used in fluorescence life time imaging (FLIM)
to reflect polarity in mitochondria. Fig. 5 shows heterogeneous
polarity distribution in mitochondria in a single cell or in different
cells, which indicates that polarity of mitochondria under different
physiology status could be different. Thus, PAHPN can be used as
an indicator for polarity in mitochondria. In future, the combina-
tion of PAHPN’s polarity-sensitivity and FLIM technique will be
further applied in order to monitor the mitochondrial micro-
environmental variations under different physiological, pharma-
cological, and toxicological conditions.
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Furthermore, PAHPN proves to be a specific mitotracker. A
colocalization imaging experiment has been performed. PAHPN
and two commercial mitrotrackers (tetramethylrhodamine
methyl ester, TMRM and mitotracker deep red) are adopted
to co-stain different cell lines including MCF-7 cells, RAW264.7
cells and COS-7 cells. As TMRM and mitotracker deep red emits
at considerably longer wavelengths than PAHPN, their different
fluorescence signals can be collected in two channels without
crosstalk. As shown in Fig. 4, in MCF-7 cells, the green
fluorescence in the PAHPN channel overlaps well with the red
fluorescence in the TMRM (or mitotracker deep red) channel,
which produces a high colocalization coefficient up to 0.92 (or
0.85 in the case of mitotracker deep red) and indicates that
Fig. 4. Colocalization imaging of MCF-7 cells costained by PAHPN and commercial mitrotracker TMRM (a–d) mitotracker deep red (e–h) for 3 min at 37 8C. (a and e)
Fluorescence image of PAHPN ( ex = 405 nm, em = 470–530 nm). (b) Fluorescence image of TMRM, ex = 559 nm, em = 600–630 nm. (c) Overlay image of a and b,
em = 655–755 nm. (g) Overlay image of e and f, colocalization coefficient 0.85. (d
l
l
l
l
colocalization coefficient 0.92. (f) Fluorescence image of mitotracker deep red,
and h) Brightfield image.
lex = 635 nm, l
Please cite this article in press as: Y. Dai, et al., A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis,
photophysical properties and cell imaging, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.020

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Y. Dai et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
5
investigations on cellular processes related to mitochondrial 231
dynamics are now being studied.
232
Acknowledgments
233
We thank National Natural Science Foundation of China (Nos. Q2234
21174022, 21376038), National Basic Research Program of China 235
(No. 2013CB733702), Key Project of the education department of 236
Sichuan Province (No. 12ZA087), and Specialized Research Fund for 237
the Doctoral Program of Higher Education (No. 20110041110009). 238
Appendix A. Supplementary data
239
Supplementary data associated with this article can be found, in 240
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.05.020. 241
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4. Conclusion
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In conclusion, PAHPN, a new two-photon mitotracker has been
efficiently synthesized through the attachment of a triphenylpho-
sphonium moiety to the naphthalimide fluorophore. PAHPN
exhibits strong fluorescence in nonpolar solvents but extremely
weak fluorescence in water, which favors the reduction of
background fluorescence from the aqueous environment outside
the mitochondrial membrane. This new molecular probe shows
reasonable two-photon excitation emission activity and presents
high-quality fluorescence images under two-photon microscopy.
PAHPN’s specificity toward mitochondria has been confirmed by
the colocalization imaging studies on three different cell lines with
two commercial mitotrackers TMRM and mitotracker red as the
references. FLIM imaging using PAHPN has mapped the different
polarity of mitochondria. The applications of PAHPN in the
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Please cite this article in press as: Y. Dai, et al., A two-photon mitotracker based on a naphthalimide fluorophore: Synthesis,
photophysical properties and cell imaging, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.020