1,8-Naphthalimide Derivative Dyes with Large Stokes Shifts for Targeting Live-Cell Mitochondria

Article abstract of DOI:10.1002/cbic.201600169

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8-naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8-naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt-4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt-4 stood out as a potential mitochondrial dye for living-cell experiments involving induced mitochondrial stress arising from the treatments because Mt-4 shows enhanced fluorescence in mitochondrial stress situations.

Full text of DOI:10.1002/cbic.201600169

Accepted Article
Title: 1,8-Naphthalimide derivative dyes targeting live cell mitochondria with large
stokes shift
Authors: Liqiang Zhang; Fengyu Su; Xiangxing Kong; Fred Lee; Steven Sher; Kevin
Day; Yanqing Tian; Deirdre R Meldrum
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To be cited as: ChemBioChem 10.1002/cbic.201600169
Link to VoR: http://dx.doi.org/10.1002/cbic.201600169
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ChemBioChem
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1
,8-Naphthalimide derivative dyes targeting live cell mitochondria
with large stokes shift
[
a]
[a]
[a]
[a]
[a]
[a]
Liqiang Zhang, Fengyu Su, Xiangxing Kong, Fred Lee, Steven Sher, Kevin Day, Yanqing
Tian*
[
a, b]
[a]
and Deirdre R. Meldrum*
Abstract: An ideal fluorescent dye for staining cell organelles should
have multiple properties including specificity, stability,
compounds with a large Stokes shift. They are attractive
fluorophores for the development of bioimaging probes with low
auto-fluorescence and low light scattering from biological
biocompatibility and a large Stokes shift. Tuneable photophysical
properties enable 1,8-naphthalimide to be an excellent fluorophore in
biomedical applications. Many naphthalimide derivatives have been
developed into drugs, sensors and other dyes. In this study, a series
of 1,8-naphthalimide derivatives targeting live cell mitochondria were
synthesized. Among these probes, Mt-4 was characterized as the
best one with highly specific mitochondrial localization, low
cytotoxicity and large Stokes shift. More importantly, Mt-4 stood out
as a potential mitochondrial dye for living cell experiments with
induced mitochondrial stress from treatments since Mt-4 has
enhanced fluorescence while undergoing mitochondrial stress.
[
6]
environments. Our lab has developed a series of fluorescent
[7]
biosensors,
including 1,8-naphthalimide derivatives as
[
8]
intracellular and extracellular pH sensors. In this study, a
series of naphthalimide derivatives (Mt-dyes) were synthesized
and functionalized with different mitochondria targeting groups,
which are TPP, pyridine and 4-dimethyaminopyridine (DMAP),
respectively (Scheme 1).
Scheme 1. Synthesis of mitochondrial dyes.
Introduction
Mitochondria play a central role in energy metabolism through
oxidative phosphorylation (OXPHOS) and lipid oxidation within
eukaryotic cells. They are also involved in many cell signaling
[1]
pathways including apoptosis.
The morphology and
abundance of mitochondria varies highly with cell type,
[
2]
proliferative state and other metabolic requirements. Mutation
in mitochondrial proteins encoded by mitochondrial DNA or
nuclear DNA will cause defects in mitochondrial function, which
[
3]
will result in serious diseases and developmental retardation.
Bound by a double membrane, mitochondria are divided into two
distinct parts: the intermembrane space and the mitochondrial
matrix. In a healthy cell, a relatively constant membrane
potential of around ca. -180 mV across the mitochondrial inner
membrane is maintained by oxidative phosphorylation and ion
[
4]
channels.
Based on these physical characteristics of
mitochondria, many fluorescent dyes and probes have been
developed to specifically stain this organelle. One of the widely
used carriers for such probes is triphenyl phosphonium (TPP), a
lipophilic cation with an excellent ability to pass through the
[
5]
mitochondrial lipid bilayer membrane.
1,8-naphthalimide derivatives are strong fluorescent
[
[
a]
b]
Dr. L. Zhang, Dr. F. Su, Dr. X. Kong, F. Lee, S. Sher, K. Day, Dr. Y.
Tian and Dr. D. R. Meldrum
Center for Biosignatures Discovery Automation, Biodesign Institute
Arizona State University
Results and Discussion
1001 S. McAlister Ave., P.O. Box 876501, Tempe, AZ 85287 (USA)
Photophysical properties of Mt-dyes
E-mail: deirdre.meldrum@asu.edu.
Dr. Y. Tian
Department of Materials Science and Engineering
South University of Science and Technology of China
No. 1088, Xueyuan Rd., Xili, Nanshan District, Shenzhen,
Guangdong, 518055 (China)
Mt-dyes have low solubility in an aqueous condition (PBS buffer)
but are completely soluble in a polar solvent, such as DMSO. To
characterize the photophysical properties of Mt-dyes, the
compounds were dissolved in DMSO and diluted with PBS
buffer by 1:500. The typical absorption and emission spectra
were collected from compound Mt-4. As shown in Figure 1, an
absorption maximum peak was seen at 408 nm, while an
E-mail: tianyq@sustc.edu.cn
Supporting information for this article is given via a link at the end of
the document.
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ChemBioChem
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emission maximum was detected at 525 nm. There is a large
Stokes shift of 117 nm with a very narrow overlap between the
absorption and the emission peak, which is one of the highly
desired characteristics for fluorescence imaging.
Mitochondrial localization
The mitochondrial localization of these compounds was
characterized with HeLa cells (Figure 3 and S2 Figure). All of
the Mt-1, Mt-4 and Mt-5 dyes, which have TPP as the
mitochondrial targeting group, showed high colocalization with
®
MitoTracker Red FM (Life Technologies, MitoRed, the same
[
10]
below) with Pearson’s correlation coefficient (PCC) about 0.9
.
The other compounds (Mt-2, 3, and 7) showed low
6
colocalization with MitoRed, i.e. Pearson’s correlation is less
than 0.7. The derivative without mitochondria targeting group,
compound 5b, aggregated in cells and could not specifically
stain mitochondria (S2 Figure). Considering the mitochondrial
localization and cytotoxicity of these mitochondrial dyes, Mt-4
was selected as the best candidate for further study.
Figure 1. Absorption and emission spectra of Mt-4. Mt-4 was dissolved into
DMSO to 2.5 mM and diluted into phosphate buffered saline (PBS) to 5.0 µM
of dyes (1:500). The UV-Vis absorption spectrum was tested from 350nm to
530nm. The emission spectrum of Mt-4 was measured under 408nm excitation.
Cytotoxicity of mitochondrial dyes
Cytotoxicity of these mitochondrial dyes derived from 1,8-
naphthalimide was assessed by MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5 diphenyltetrazoliumbromide) assay, which determines
[
9]
mitochondrial activity . The HeLa cell line was used as a typical
cell line to test toxicity. Cells were incubated with 1.0 µM
mitochondrial dyes for 3, 6, 12 and 24 hours, respectively.
Compared to negative control (DMSO solvent only) and other
derivatives, Mt-1 and Mt-5, both of which have six carbon chains
and TPP as the targeting group, showed strong cytotoxicity in 12
hour and longer incubation, while the Mt-4 did not show much
toxicity to cells (Figure 2). The only difference between Mt-4 and
Mt-5 is the length of carbon chain connecting the fluorophore
and mitochondrial targeting group TPP. The mechanism of the
toxicity will be studied further in future research.
Figure 3. Fluorescent images of HeLa cells co-stained with mitochondrial dyes.
(
A) Mt-4(1 µM) and (B) MitoTracker Red FM (MitoRed, 0.2 µM). (C) is the
merged image from A and B. Excitation wavelength: 405 nm (for Mt-4) and
61 nm (for MitoRed).
5
Figure 4. Signal loss of fluorescent emission of Mt-4(green) and MitoRed (red)
dyes during mitochondrial co-staining. A, relative fluorescence signal (I/I ) after
adding Mt-4 to cells which were pre-stained with MitoRed; B, relative
fluorescence signal (I/I ) after adding MitoRed to cells pre-stained with Mt-4; C,
0
0
Figure 2. Cytotoxicity of mitochondrial dyes. The compound 5b with six carbon
fluorescent emission from cells pre-stained with Mt-4 or MitoRed; D,
fluorescent images when cells were stained by additional dyes (Mt-4 in upper
panel and MtRed in lower panel) for 0 and 30 minutes.
chain (scheme 1, n=6) is also tested in this assay.
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Figure 5. Fluorescence from mitochondrial dyes is affected by CCCP
added to cells. A, relative fluorescence from Mt-4 or MitoRed. B, cell images
taken by con-focal microscopy. a) MitoRed; b) Mt-4.
During the co-staining of Mt-4 and MitoRed, one interesting
phenomena was observed. After Mt-4 (1.0 µM) was added into
the medium with cells, which were first stained with MitoRed (0.2
µM), a quick decrease of fluorescence from MitoRed (Em650nm
)
Discussion
was detected (Figure 4A, D and S3 Figure A). The green
fluorescence from Mt-4 was saturated in five minutes. On the
contrary, if cells were stained with Mt-4 first, the red
fluorescence from MitoRed was saturated for about ten minutes
after highly concentrated MitoRed (final concentration is 1.0 µM)
was added, while only ~10% decrease of green fluorescence
from pre-stained Mt-4 was observed (Figure 4B, D and S3
Figure B). As controls, cells were stained only using either Mt-4
With the need for increasing the sensitivity of biochemical
assays, fluorescence is one of the most powerful tools which
has been widely used in biomedical imaging and other related
fields. An ideal fluorophore should have a large Stokes shift.
Otherwise, it can cause self-quenching and false positives
[
11]
caused by excitation light and scattered light.
Despite a
cornucopia of options which are commercially available,
researchers still have been spending much of their efforts
towards the development of new dyes with specific excitation or
(
1.0 µM) or MitoRed (0.2 µM), no obvious fluorescence intensity
decrease was observed during the same experimental time
frame and similar experimental conditions (Figure 4C and S4
Figure).
[
12]
emission requirements.
In this study, eight compounds were biologically characterized
and only Mt-4 stood out as the best candidate with good
mitochondrial localization (Figure 3) and biocompatible
properties (Figure 2). The data collected from a co-staining
assay (Figure 4 and S4 Figure) indicates that the fluorescence
signal loss of the MtRed is not due to photobleaching during
microscopy imaging. The possible “quenching effect” of MitoRed
by Mt-4, which happens between mitochondrial dyes and some
Fluorescence from mitochondrial dyes is affected by
membrane potential
In this study, HeLa cells were stained with either MitoRed or Mt-
4
before cells were treated with 10 µM of CCCP. As shown in
Figure 5, there is about a 25% signal loss from MitoRed in one
hour of CCCP treatment. On the contrary, the fluorescence of
Mt-4 increased about 40% in the first 40 minutes of treatment
followed by slowly recovering with the longer incubation (Figure
[13]
mitochondria targeting compounds, was also excluded by an
in vitro assay. Various concentrations of the mixtures with Mt-4
and MitoRed were mixed in DMSO for the measurements of
their emission properties. Results showed that the
concentrations of MitoRed or Mt-4 did not affect the
fluorescence intensities of the other dye, i.e. Mt-4 or MitoRed
5A). According to the microscopy images, except for the
morphological changes of the mitochondria, no obvious
mitochondrial dyes leakage was observed in HeLa cells stained
by either Mt-4 or MitoRed (Figure 5B). The possible reason for
the fast increase of Mt-4 fluorescence after CCCP treatment is
the fragmentation of mitochondria caused by treatments, which
may change the microenvironmental distribution of Mt-4.
(
S5 Figure). However, solvents with different polarities affect the
fluorescent emission of Mt-4 and MitoRed in different patterns
S6 Figure). Therefore, the possible reason for the quick decay
(
of fluorescence from MitoRed by Mt-4 is that the local
microenvironmental change of MitoRed is caused by the fast
mitochondrial accumulation of Mt-4, while accumulation of
MitoRed could not affect the distribution of Mt-4 in the same way.
As mentioned above, the driving force for Mt-4 with a positive
charge targeting mitochondria is the membrane potential of ca. -
180 mV across the mitochondrial inner membrane. But this
value and morphology of mitochondria are dynamically changed
[14]
when cells are under stress. So the stability of fluorescence
from dyes staining mitochondria is critical to the live cell imaging
under treatments causing vibration of mitochondrial membrane
potential (MMP). Carbonyl cyanide m-chlorophenylhydrazone
(
CCCP) is traditionally used as an OXPHOS uncoupling agent
working as a proton transmembrane carrier which results in the
[
15]
quick reduction of mitochondrial inner membrane potential. It
has been reported that the MitoRed lost its specificity to
mitochondria when the cells were pre-treated with 10 µM of
CCCP and the sensitivity and specificity to mitochondria through
[
16]
the observation of a decrease in fluorescence intensities. It
was reported that CCCP leads to the fragmentation of
mitochondria and inhibits the mitochondrial fusion, with the loss
[
14]
of membrane potential it induces. Similar to the decrease of
MitoRed fluorescence during CCCP treatment, we think the
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ChemBioChem
10.1002/cbic.201600169
FULL PAPER
increase of fluorescence from Mt-4 is the result of the
microenvironmental changes of dyes induced by CCCP in a
unknown mechanism.
Mitochondrial dyes were synthesized according to the procedure shown
in Scheme 1. The crude products were purified by silica gel column
1
[
17]
chromatography. The structures of compounds were confirmed by H and
13
C NMR spectroscopy and mass spectra. The details of the synthesis of
each compound are given in the results part.
Conclusions
Synthesis of compound 6a (Mt-4)
In summary, a series of naphthalimide derived mitochondrial
dyes were synthesized and tested. Mt-4 has been identified as
the best one among the compounds we prepared due to its large
Stokes shifts, low toxicity, and unique mitochondrial staining
properties. It is expected to be a useful imaging dye for
mitochondria-related research either at the bulk cell level or
325 mg (1 mmol) of 9-(2-Hydroxyethyl)-4-(N-piperidino)-1,8-
naphthalimide (compound 2a, n=2) was dissolved into 20 mL of ice-cold
anhydrous tetrahydrofuran (THF) containing 303.6 mg of trimethylamine
(
Et
3
N). After dropwise addition of 10 mL of anhydrous THF containing
229 mg (2 mmol) of methanesulfonyl chloride, the reaction mixture was
vigorously stirred overnight at room temperature. Solvent was removed
under reduced pressure. The crude product was dissolved into 100 mL of
dichloromethane and was washed with water three times. The organic
layer was dried with sodium sulfate and the solvent was removed under
vacuum. Without further purification, the product (compound 3a, n=2)
was dissolved into 10 mL of anhydrous acetonitrile with 310 mg (3 mmol)
sodium bromide (NaBr). The mixture was incubated at room temperature
under vigorous stirring for 24 hours. 100 mL of dichloromethane was
added to reaction mixture followed by washing two times with water and
washing one time with brine. The organic phase was dried by sodium
sulfate and the solvent was removed under reduced pressure. The
product was purified by silica gel chromatography using 85:15 of hexane-
[
18]
single cell level.
Experimental Section
Reagents and instruments
All chemicals were purchased and used without further purification.
MitoTracker® Red FM was ordered from Life Technologies (Carlsbad,
CA).
compound 1a, n=2) and N-(6-hydroxyhexyl)-4-bromine-1,8-naphthalimide
Figure 1, compound 1b, n=6), 9-(2-hydroxyethyl)-4-(N-piperidino)-1,8-
naphthalimide (compound 2a, n=2) and 9-(6-hydroxyhexyl)-4-(N-
piperidino)-1,8-naphthalimide (compound 2b, n=6) were prepared
N-(2-hydroxyethyl)-4-bromine-1,8-naphthalimide
(Figure
1,
ethyl acetate to get 272 mg of 2-(2-bromoethyl)-6-(piperidin-1-yl)-1H-
1
benzo[de]isoquinoline-1,3(2H)-dione (compound 5a, n=2), yield 70%.
H
(
NMR (400 MHz, CDCl3, δ ppm): 1.73 (m, 2H), 1.86 (m, 4H), 3.24(t, 4H),
3
1
.63 (t, 2H), 4.57 (t, 2H), 7.14 (d, 1H), 7.65 (t, 1H), 8.39 (d, 1H), 8.50 (d,
H), 8.58 (d, 1H).
[
8a, 19]
according to known procedure
.
1
95 mg (0.5 mmol) of compound 5a (n=2) was dissolved into 10mL
1
A Varian liquid-state NMR operated at 400 MHz was used for H and
C
ethanol with 787 mg (3 mmol) of triphenylphosphine (TPP). The mixture
was refluxed for 24 hours. The solvent was removed by vacuum and the
product was purified by silica gel chromatography using 93:7
dichloromethane-methane as eluent to get 168 mg orange product, yield
2%. H NMR (400MHz, CDCl3, δ ppm): 1.72 (m, 2H), 1.86 (m, 4H),
.24(t, 4H), 4.10 (m, 2H), 4.60 (t, 2H), 7.11 (d, 1H), 7.62-7.72 (m, 10H),
.83-7.88(m, 6H), 8.35-8.37 (m, 2H), 8.43 (d, 1H). C NMR (100 MHz,
CDCl3, δ ppm): 164.4, 163.8, 158.2, 135.4, 133.8, 133.7, 133.5, 131.7,
30.8, 130.7, 130.2, 126.2, 125.5, 122.3, 118.3, 117.5, 114.8, 114.7,
10.2, 54.7, 34.5, 26.3, 24.4, 22.3, 21.9) (S1 Fig). MS (MOLDI-TOF): m/z
1
3
NMR spectra measurements. An Applied Biosystems DE-STR
MALDI-TOF Mass Spec was used to measure the mass spectra of the
intermediates and the final product. A Shimadzu UV-3600 UV-Vis-NIR
spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) was
used for UV-Vis absorption spectra measurements. A Shimadzu RF-
301 spectrofluorophotometer was used for fluorescence measurements.
Confocal microscope (Nikon, TE2000E, Melville, NY) was used for cell
imaging.
1
5
3
7
5
13
1
1
Experimental condition for cell culture, imaging, toxicity, and
colocalization study.
+
+
calcd C37H34N2O2P (M) 569.66, found 569.62.
Synthesis of compound 4a (Mt-1)
HeLa cells (American Type Culture Collection, ATCC, Manassas, VA)
were cultured in Eagle’s minimum essential medium (EMEM)
supplemented with 10% fetal bovine serum, and incubated at 37 °C in
381 mg (1 mmol) of 9-(6-Hydroxyhexyl)-4-(N-piperidino)-1,8-
naphthalimide (Scheme 1, compound 2b, n=6) was dissolved into 20 mL
of ice-cold anhydrous tetrahydrofuran (THF) containing 303.6 mg of
trimethylamine (Et3N). After dropwise addition of 10 mL of anhydrous
THF containing 229 mg (2 mmol) of methanesulfonyl chloride, the
reaction mixture was vigorously stirred overnight at room temperature.
Solvent was removed under reduced pressure. The crude product was
dissolved into 100 mL of dichloromethane and was washed with water for
three times. The organic phase was dried with sodium sulfate and the
solvent was removed under vacuum. Without further purification, the
product (compound 3, n=3) was dissolved into 30 mL of anhydrous
acetonitrile with 1.32 g (5 mmol) of TPP. The mixture was refluxed for 24
hours. After removal of solvent by vacuum rotary evaporation, the
product was purified by silica gel chromatography using 93:7
5
1
% CO2 atmosphere. Cells were then seeded onto 96 well plates at
0,000 cells per well, and incubated for 1 day at 37 °C. For the
colocalization assay, cells were pre-incubated with MitoTracker® Red FM
for half hours. Mitochondrial dyes were dissolved into DMSO and added
to medium to get the final concentration of dyes at 1.0 µM. Confocal
images were taken five minutes after adding the mitochondrial dyes. For
the kinetic imaging assay, confocal imaging was started immediately
when the dyes were added.
For the cell toxicity assay, cells were incubated with the sensor for 6,
12 and 24 hours before the assay was performed with the cytotoxicity
MTT kit from Promega (CellTiter 96® Non‐Radioactive Cell Proliferation
Assay) following the detailed procedure from the company.
dichloromethane-methane as eluent to get 210 mg orange product, yield
1
2
3
7
9.1%. H NMR (CDCl3, δ ppm): 1.72 (m, 2H), 1.86 (m, 4H), 2.71 (s, 3H),
.21(t, 4H), 3.77 (m, 2H), 4.06 (t, 4H), 7.13 (d, 1H), 7.23(d, 1H), 7.62-
.70 (m, 7H), 7.74-7.85(m, 9H), 8.35 (d, 1H), 8.43 (d, 1H), 8.49 (d, 1H).
Synthesis of mitochondrial dyes
+
+
MS (MOLDI-TOF): m/z calcd C41H42N2O2P (M) 625.77, found 625.72.
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10.1002/cbic.201600169
FULL PAPER
Synthesis of compound 4b (Mt-2)
This work was supported by the NIH National Human Genome
Research Institute (NHGRI), Centers of Excellence in Genomic
Science, grant number 5 P50 HG002360, and the NIH Common
By a procedure similar to the synthesis of Mt-1, 381 mg (1 mmol) of
compound 2b with 6 carbons chain (n=6) and 367 mg (3 mmol, 3 times of
starting compound 2b) of 4-dimethylaminopyridine (DMAP) were applied
Fund LINCS program, grant number
(Professor Deirdre R. Meldrum, PI).
5 U01 CA164250
1
to the reaction to produce 260 mg pure product, yield 45%. H NMR
(CDCl3, δ ppm): 1.72 (m, 2H), 1.86 (m, 4H), 2.71 (s, 3H), 3.20(s, 4H),
3
1
.22(t, 6H), 4.08 (m, 2H), 4.22 (t, 2H), 6.97 (t, 2H), 7.12 (d, 1H), 7.23(d,
H), 8.30-8.35(m, 3H), 8.43 (d, 1H), 8.50 (d, 1H). MS (MOLDI-TOF): m/z
Keywords: naphthalimide • mitochondria • stokes shift • single
live cell
+
+
calcd C30H37N4O2 (M) 485.65, found 485.60.
[
[
1] D. R. Green, J. C. Reed, Science 1998, 281, 1309-1312.
Synthesis of compound 4c (Mt-3)
2] a) S. A. Detmer, D. C. Chan, Nature reviews. Molecular cell biology 2007,
8
, 870-879; b) M. Karbowski, R. J. Youle, Cell death and differentiation
By a procedure similar to the synthesis of Mt-2, 381 mg (1 mmol) of
compound 2b with 6 carbons chain (n=6) and 237 mg (3 mmol, 3 times of
starting compound 2b) of pyridine were applied to the reaction to produce
2003, 10, 870-880; c) A. P. Leonard, R. B. Cameron, J. L. Speiser, B. J.
Wolf, Y. K. Peterson, R. G. Schnellmann, C. C. Beeson, B. Rohrer,
Biochimica et biophysica acta 2015, 1853, 348-360.
1
2
1
2
8
26 mg pure product, yield 40%. H NMR (CDCl3, δ ppm): 1.71 (m, 2H),
.84 (m, 4H), 2.07 (t, 2H), 2.71 (s, 3H), 3.20(t, 4H), 3.22(s, 6H), 4.07 (t,
H), 5.01 (t, 2H), 7.12 (d, 1H), 7.64(t, 1H), 8.10(t, 2H), 8.34 (d, 1H), 8.47-
[
3] a) A. H. Schapira, Lancet 2006, 368, 70-82; b) D. C. Wallace, Science
1999, 283, 1482-1488.
+
+
[4] a) J. R. Casey, S. Grinstein, J. Orlowski, Nature reviews. Molecular cell
biology 2010, 11, 50-61; b) L. B. Chen, Annual review of cell biology 1988,
.50 (m, 2H). MS (MOLDI-TOF): m/z calcd C28H32N3O2 (M) 442.58,
found 442.51.
4, 155-181.
[
5] a) B. A. D. Neto, J. R. Correa, R. G. Silva, Rsc Adv 2013, 3, 5291-5301; b)
O. A. de Carvalh, R. F. Guimaraes, N. C. Silva, A. R. Gillespie, R. A. T.
Gomes, C. R. Silva, A. P. F. de Carvalho, Remote Sens-Basel 2013, 5,
Synthesis of compound 6b (Mt-5)
By a procedure similar to the synthesis of Mt-4, 760 mg (2 mmol) of 9-(2-
Hydroxyhexyl)-4-(N-piperidino)-1,8-naphthalimide (compound 2b, n=6)
and 460 mg (4 mmol) of methanesulfonyl chloride were used to
synthesize compound 5b (scheme 1, n=6), 2-(6-bromohexyl)-6-(piperidin-
2763-2794; c) M. I. Rosa, B. R. Silva, P. S. Pires, F. R. Silva, N. C. Silva,
F. R. Silva, S. L. Souza, K. Madeira, A. P. Panatto, L. R. Medeiros, Eur J
Obstet Gyn R B 2013, 167, 132-136; d) C. A. L. Oliveira, N. C. Silva, C. L.
Sabat, R. M. F. Lima, Comput Inform 2013, 32, 225-250.
1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to produce 650 mg of pure
1
[6] S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy, G. M. Tocci, L. J.
Gillespie, D. O. Frimannsson, J. M. Kelly, T. Gunnlaugsson, Chemical
Society reviews 2013, 42, 1601-1618.
product in yellow powder state, yield 74%. H NMR (CDCl3, δ ppm):
1
1
.71 (m, 2H), 1.85 (m, 4H), 3.21(t, 4H), 3.83(t, 2H), 4.15 (t, 2H), 7.12 (d,
H), 7.64(t, 1H), 8.38 (d, 1H), 8.50 (d, 1H), 8.58 (d, 1H).
[
7] a) H. Lu, Y. Jin, Y. Tian, W. Zhang, M. R. Holl, D. R. Meldrum, J Mater
Chem 2011, 21, 19293-19301; b) X. Zhou, F. Su, Y. Tian, C. Youngbull, R.
H. Johnson, D. R. Meldrum, Journal of the American Chemical Society
2011, 133, 18530-18533; c) X. Zhou, F. Su, H. Lu, P. Senechal-Willis, Y.
Tian, R. Johnson, D. R. Meldrum, Biomaterials 2012, 33, 171-180; d) F.
Su, R. Alam, Q. Mei, Y. Tian, C. Youngbull, R. H. Johnson, D. R. Meldrum,
Plos One 2012, 7; e) L. Zhang, F. Su, S. Buizer, H. Lu, W. Gao, Y. Tian, D.
Meldrum, Biomaterials 2013, 34, 9779-9788; f) L. Zhang, F. Su, S. Buizer,
X. Kong, F. Lee, K. Day, Y. Tian, D. R. Meldrum, Chem Commun 2014,
221 mg (0.5 mmol) of compound 5b and 787 mg (3 mmol) of TPP
were used to synthesize Mt-5 and got 230 mg of pure product, yield 65%.
1
H NMR (CDCl3, δ ppm): 1.72 (m, 2H), 1.86 (m, 4H), 3.21(t, 4H), 3.77 (m,
2
7
H), 4.06 (t, 4H), 7.13 (d, 1H), 7.23(d, 1H), 7.62-7.70 (m, 7H), 7.74-
.85(m, 9H), 8.35 (d, 1H), 8.43 (d, 1H), 8.49 (d, 1H). MS (MOLDI-TOF):
+
+
m/z calcd C41H42N2O2P (M) 625.77, found 625.71.
Synthesis of compound 6c (Mt-6)
50, 6920-6922.
[
8] a) Y. Tian, F. Su, W. Weber, V. Nandakumar, B. R. Shumway, Y. Jin, X.
Zhou, M. R. Holl, R. H. Johnson, D. R. Meldrum, Biomaterials 2010, 31,
7411-7422; b) Y. Tian, B. R. Shumway, A. C. Youngbull, Y. Li, A. K. Y.
Jen, R. H. Johnson, D. R. Meldrum, Sensor Actuat B-Chem 2010, 147,
By a procedure similar to the synthesis of Mt-5, 221 mg (0.5 mmol) of
compound 5b and 183 mg (1.5 mmol) of DMAP were used to synthesize
Mt-6 to produce 249 mg of pure product, yield 88%. H NMR (CDCl3, δ
1
ppm): 1.72 (m, 2H), 1.86 (m, 4H), 3.20(s, 4H), 3.22(t, 6H), 4.08 (m, 2H),
714-722.
4
.22 (t, 2H), 6.97 (t, 2H), 7.12 (d, 1H), 7.23(d, 1H), 8.30-8.35(m, 3H),
+
[9] a) J. van Meerloo, G. J. Kaspers, J. Cloos, Methods in molecular biology
2011, 731, 237-245; b) T. L. Riss, R. A. Moravec, A. L. Niles, H. A. Benink,
T. J. Worzella, L. Minor, in Assay Guidance Manual (Eds.: G. S.
Sittampalam, N. Gal-Edd, M. Arkin, D. Auld, C. Austin, B. Bejcek, M.
Glicksman, J. Inglese, V. Lemmon, Z. Li, J. McGee, O. McManus, L.
Minor, A. Napper, T. Riss, O. J. Trask, J. Weidner), Bethesda (MD), 2004.
8.43 (d, 1H), 8.50 (d, 1H). MS (MOLDI-TOF): m/z calcd C30H37N4O2
+
(M) 485.65, found 485.60.
Synthesis of compound 6d (Mt-7).
By a procedure similar to the synthesis of Mt-5, 221 mg (0.5 mmol) of
compound 5b and 120 mg (1.5 mmol) of pyridine were used to
[
10] K. W. Dunn, M. M. Kamocka, J. H. McDonald, American journal of
physiology. Cell physiology 2011, 300, C723-742.
1
synthesize Mt-7 to produce 175 mg of pure product, yield 67%. H NMR
[
11] a) D. L. Andrews, A. A. Demidov, An introduction to laser spectroscopy,
(CDCl3, δ ppm): 1.71 (m, 2H), 1.84 (m, 4H), 2.07 (t, 2H), 3.20(t, 4H),
2nd ed., Kluwer Academic/Plenum Publishers, New York, 2002; b) J. A.
3
8
.22(s, 6H), 4.07 (t, 2H), 5.01 (t, 2H), 7.12 (d, 1H), 7.64(t, 1H), 8.10(t, 2H),
.34 (d, 1H), 8.47-8.50 (m, 2H). MS (MOLDI-TOF): m/z calcd
Richardson, T. Morgan, M. Andreou, T. Brown, The Analyst 2013, 138,
3626-3628.
+
+
C28H32N3O2 (M) 442.58, found 442.50.
[
12] a) A. C. Benniston, T. P. Winstanley, H. Lemmetyinen, N. V. Tkachenko,
R. W. Harrington, C. Wills, Organic letters 2012, 14, 1374-1377; b) X.
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American Chemical Society 2005, 127, 4170-4171; c) J. F. Araneda, W. E.
Piers, B. Heyne, M. Parvez, R. McDonald, Angewandte Chemie 2011, 50,
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ChemBioChem
10.1002/cbic.201600169
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Entry for the Table of Contents
FULL PAPER
A series of 1,8-naphthalimide
derivatives targeting live cell
mitochondria were synthesized.
Among these probes, Mt-4 is the
best one with highly specific
Liqiang Zhang, Fengyu Su, Xiangxing
Kong, Fred Lee, Steven Sher, Kevin
Day, Yanqing Tian* and Deirdre R.
Meldrum*
Page No. – Page No.
mitochondrial localization, low
cytotoxicity and large Stokes shift. It
is a potential mitochondrial dye for
living cell experiments with induced
mitochondrial stress from treatments.
Title: 1,8-Naphthalimide derivative
dyes targeting live cell mitochondria
with large stokes shift
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