A novel pH "off-on" fluorescent probe for lysosome imaging

Article abstract of DOI:10.1039/c3ra41898g

In the present paper, a new pH sensitive fluorescent probe based on a 4-acylated naphthalimide fluorophore was well designed and synthesized. It has a high specificity for lysosomes over other cell organelles. The intrinsic high signal to noise ratio in cell imaging, broad Stokes shift and practical fluorescence quantum yield of this probe will ensure its prominent position in the intracellular lysosome targeting and imaging toolbox.

Full text of DOI:10.1039/c3ra41898g

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A novel pH ‘‘off–on’’ fluorescent probe for lysosome
imaging3
Cite this: DOI: 10.1039/c3ra41898g
Laizhong Chen, Jing Li, Zhenzhen Liu, Zhao Ma, Wei Zhang, Lupei Du, Wenfang Xu,
Hao Fang and Minyong Li*
In the present paper, a new pH sensitive fluorescent probe based on a 4-acylated naphthalimide
fluorophore was well designed and synthesized. It has a high specificity for lysosomes over other cell
organelles. The intrinsic high signal to noise ratio in cell imaging, broad Stokes shift and practical
fluorescence quantum yield of this probe will ensure its prominent position in the intracellular lysosome
targeting and imaging toolbox.
Received 18th April 2013,
Accepted 31st May 2013
DOI: 10.1039/c3ra41898g
www.rsc.org/advances
probe molecule can cause a high background signal. In
comparison, Lyso Tracker Green (LT Green, 2, Fig. 1), a pH
Introduction
Lysosomes are acidic cytoplasmic membrane-bound orga-
nelles that degrade macromolecules and cell components by
using interior hydrolytic enzymes.1,2 They comprehensively
participate in many physiological processes, such as choles-
terol homeostasis, plasma membrane repair, bone and tissue
remodelling, pathogen defence, cell death and cell signalling.3
They also have indispensable capacities in oncogenic activa-
tion and cancer progression.4 Therefore, in view of the
significant biological function of lysosomes, it is worthwhile
to formulate probes to target and label lysosomes.
Lysosomes are present in all eukaryotic cell types except
erythrocytes.1 However, there are many other complicated
organelles in living cells, such as the Golgi complex and
mitochondria; therefore it is not easy to differentiate lyso-
somes from other organelles. Lysosomes can be visualized by
immunofluorescence staining using antibodies against a
certain membrane protein.5 Nevertheless, these immunologi-
cal methods are generally expensive and sophisticated. In
contrast, small fluorescent probe based imaging is an
appealing approach since it unusually presents real-time
characteristics, adequate sensitivities and high spatiotemporal
resolutions.6–8 As the pH in lysosomes (4.5–5.0) is more acidic
than in the cytoplasm (pH 7–7.3), a lysosome specific
fluorescent probe often includes a fluorophore and a basic
side chain to allow the accumulation of the probe in acidic
vesicles upon protonation.9 For example, Lyso Tracker Red (LT
Red, 1, Fig. 1)10 is one of the commonly-used commercial
lysosome probes, which has a weak basic side chain that can
accumulate in acidic lysosomes. Nonetheless, the external
sensitive probe, exhibits weak fluorescence in neutral cyto-
plasm but emits bright fluorescent light in acidic lysosomes,
which can highly escalate the signal to noise ratio.9 However,
this BODIPY-based probe has a short Stokes shift (,20 nm)
and is often unstable at room temperature.
Recently, a pH sensitive naphthalimide derivative (SM1, 3,
Fig. 1) was used to detect lysosomes since it can exhibit a large
Stokes shift, has a minimized susceptibility to interference
from metal ions, and a high photostability.11 Even so, the
quantum yield of SM1 is still relatively low, and its ester group
is unstable in the acidic environment of the lysosome. Herein,
we report a new pH sensitive 4-acylated naphthalimide-based
lysosome probe, which has a large Stokes shift, a good
quantum yield and a promising specificity.
Result and discussion
As depicted in Scheme 1, to enhance the quantum yield of the
naphthalimide derivative, we chose 4-acylated 1,8-naphthali-
mide as the fluorescent reporter based on the literature.12 D-
Phe–L-Phe was used to regulate the pKa and partition coefficient
of the probe, as it has a wide application in drug design13 and
demonstrates the advantages of using peptidomimetics in
intracellular pH sensors.14,15 Unlike SM1 and other naphthali-
mide based pH probes, N,N-dimethylethylamine was selected as
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE),
School of Pharmacy, Shandong University, Jinan, Shandong 250012, China.
E-mail: mli@sdu.edu.cn; Fax: +86-531-8838-2076; Tel: +86-531-8838-2076
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
Fig. 1 The structures of LT Red (1), LT Green (2) and SM1 (3).
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Scheme 1 A plausible detection mechanism for the LTP probe (4).
the PET (photoinduced electron transfer) donor for its
acidotropic property towards lysosomes. The intramolecular
PET quenching of the probe would result in weak fluorescence
Fig. 2 Absorption (red) and emission (black) spectra of LTP (7 mM).
in
a
neutral environment. After the acidification of
N,N-dimethylethylamine in a low pH environment, the PET
effect would be weakened, and the fluorescence would increase.
The probe can be efficiently synthesized in six steps without
column chromatography purification (Scheme 2). Briefly, L-Phe
was protected with Fmoc–OSu to afford compound 7, which was
reacted with thionyl chloride to obtain compound 8. Compound
9 was synthesized according to the literature.16 Condensation of
8 and 9 afforded the amide 10, which was deprotected in a 20%
piperidine–DMF solution to produce compound 11.
Condensation of 11 and Boc–D-Phe (12) with EDCI and HOBt
produced compound 13. Finally, 13 was deprotected with HCl–
AcOEt and then alkalized to generate LTP (4).
LTP was initially evaluated for its spectroscopic properties
under near physiological conditions (Britton–Robinson buffer,
pH = 7.4). The probe itself demonstrates absorption and
emission spectra at 350 nm and 465 nm, respectively, in
Britton–Robinson buffer (pH = 7.4). As expected, the Stokes
shift of LTP was 115 nm, which is 5–10 times longer than that
of BODIPY-based probes (Fig. 2). The excitation wavelength is
in the ultraviolet band, which can avoid the disturbance of the
excitation by natural light. Moreover, the fluorescence quan-
tum yield of LTP was 0.89, which is higher than that of 4-NH2
naphthalimide (0.11, 9 in Scheme 2) and LT Red (0.07) in
and basic conditions. However, the emission from the solution
significantly increased as the pH decreased to acidic levels.
The maximum fluorescence intensity appeared when the pH
was lower than 3.7. This fluorescence intensity change may be
attributed to the above-mentioned PET effect as illustrated in
Scheme 1. As the pH in lysosomes (4.5–5.0) is more acidic than
in the cytoplasm (pH 7–7.3),17 the appropriate pKa of LTP was
6.48, which is suitable for detecting in the pH range of 7.3–4.5,
which includes the cytoplasm and the lysosomes, in which the
fluorescence intensity of LTP increases 3.3 times. This
evidence indicates that LTP can be employed as a pH-tunable
probe for lysosome imaging.
The confocal fluorescence microscopy results show that
LTP can readily penetrate living ES-2 cells. To confirm that LTP
is able to recognize lysosomes located in the cytoplasm, ES-2
cells were co-stained with commercial markers, including Lyso
Tracker Red (LT Red), Mito Tracker Green (MT Green) and
Britton–Robinson buffer solution (pH
important for an ideal imaging probe.
= 4.5), which is
The pH response of LTP was also thoroughly examined. As
shown in Fig. 3, weak fluorescence was observed under neutral
Fig. 3 (A) The fluorescence change of LTP in Britton–Robinson buffer at
different pH values; (B) the fluorescence intensity at 460 nm depending on
different pH values, lex = 360 nm.
Scheme 2 The synthesis of the LTP probe.
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Fig. 5 Co-staining of 3.7% formaldehyde fixed ES-2 cells with LTP (7 mM) and LT
Red (70 nM). Row A shows images that were taken immediately after
incubation. Row B shows snapshot images after washing the unbound probes.
(a) Rows A and B show bright-field images. (b) Rows A and B show cells stained
by LTP, lex = 405 nm; (c) rows A and B show cells stained by LT Red, lex = 555
nm; (d) rows A and B show merged images. All images were taken under a 636
oil immersion objective.
Fig. 4 Colocalization images of ES-2 cells incubated with LTP and the three
commercial trackers. The four images in each row show: (a) bright-field images;
(b) cells stained by LTP, (lex = 405 nm); (c) cells stained by LT Red (lex = 555 nm),
GT Red (lex = 555 nm) and MT Green (lex = 488 nm) in rows A, B and C,
respectively; (d) merged images. All images were taken under a 636 oil
immersion objective.
ophore and the plausible physicochemical characteristic of
peptidomimetic D-Phe–L-Phe, a novel tailor-made pH ‘‘off–on’’
lysosomal imaging probe was well designed and synthesized in
the current report. After careful experimental determination,
our probe was found to have beneficial specificity for
lysosomes over other cell organelles. Moreover, this fluores-
cent probe has improved characteristics, including a high
signal to noise ratio, a large Stokes shift, a good fluorescence
quantum yield and an acceptable feasibility in immunofluor-
escence co-staining, when compared with the commercial
lysosome probe LT Red. In addition, the probe has the
advantage of a convenient synthesis from readily available
inexpensive starting materials. We expect that these stunning
results may eventually lead to a brand new tool kit for
intracellular lysosome targeting and imaging.
Golgi Tracker Red (GT Red). Cellular confocal images demon-
strated that the localization of LTP in the cells was nearly
identical to that of LT Red (Fig. 4A). However, for golgiosomal
and mitochondrial colocalization, the localization of LTP did
not overlap with that of the typical commercial GT Red and MT
Green (Fig. 4B and 4C). The colocalization coefficient was
calculated using ImageJ software,18 in which Pearson’s
method was applied to evaluate the colocalization of LTP
relative to the commercial probes. The colocalization coeffi-
cient (0.90) of LTP with LT Red was much higher than that
with GT Red (0.43) and MT Green (0.41), respectively, thus
revealing the lysosomal specificity of LTP. Furthermore,
considering that LTP is a pH ‘‘off–on’’ probe, the signal to
noise ratio of LTP in lysosome imaging was much higher than
that of LT Red (10.5 versus 2.7, Fig. 4Ab and 4Ac, calculated by
the ImageJ 1.46 program using Fig. 4Ad as the reference).
Cell fixation is a substantial procedure in immunofluores-
cence staining.19 However, most of the LT Red was observed to
disappear when the fixed ES-2 cells were washed after staining.
In contrast, the majority of the LTP molecules remained in the
lysosomes after washing (Fig. 5), which indicates that it is
more feasible to use LTP in immunofluorescence co-staining
rather than LT Red.
Experimental
1. Materials and instruments
Lyso Tracker Red, Mito Tracker Green and Golgi Tracker Red
were purchased from the Beyotime Institute of Biotechnology.
ES-2 and PC-3 cells were purchased from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). Buffer
reagents were purchased from Aldrich and Acros and were
used without purification. Water used for the fluorescence
studies was doubly distilled and further purified with a Mill-Q
filtration system. Absorption spectra were recorded using a
Shimadzu UV-1700 UV-visible spectrometer. Fluorescence
spectra were recorded using Varioskan (Thermo Electron
Corporation). 1H NMR and 13C NMR were recorded on
Bruker 300 NMR, 400 NMR and 600 NMR spectrometers.
Mass spectra were obtained using the analytical and mass
spectrometry facilities at Shandong University. Specific rota-
tion was tested using Anton Paar MCP200. HPLC was
performed with Agilent 1260. Confocal microscopy was
recorded using a Zeiss LSM 700.
To gain a preliminary understanding of the lysosomal
specificity for LTP in other cell lines, PC-3 cells were also co-
stained with LT Red and LTP. The colocalization images (Fig.
S1, ESI ) and colocalization coefficient (0.91) indicate that LTP
3
can also specifically label lysosomes in other cells such as PC-
3.
Conclusions
In summary, by taking advantage of the promising spectro-
scopic properties of the 4-acylated 1,8-naphthalimide fluor-
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concentrated to afford a yellowish powder, 0.034 g, yield =
85.0%. HPLC purity 94.4%. mp: 99.5–101.1 uC. [a]2D0 = 271.65 (c
= 0.13, MeOH). 1H NMR (600 MHz, MeOD) d 8.49 (d, J = 6.6 Hz,
1H), 8.43 (d, J = 11.4 Hz, 1H), 7.96 (d, J = 9.6 Hz, 2H), 7.68 (t, J =
7.8 Hz, 1H), 7.32–7.38 (m, 4H), 7.26–7.29 (m, 3H), 7.20 (d, J =
7.2 Hz, 1H), 7.17 (d, J = 7.2 Hz, 2H), 4.91 (t, J = 7.2 Hz, 1H), 4.30
(t, J = 7.2 Hz, 2H), 3.69 (t, J = 7.2 Hz, 1H), 3.15 (dd, J = 13.2, 8.4
Hz, 1H), 2.94–3.03 (m, 2H), 2.84 (dd, J = 13.2, 7.2 Hz, 1H), 2.71
(t, J = 7.2 Hz, 2H), 2.38 (s, 6H). 13C NMR (150 MHz, MeOD) d
139.0, 137.2, 136.6, 131.0, 130.8, 129.2, 129.1, 128.8, 128.5,
128.4, 128.1, 126.8, 126.4, 126.3, 125.1, 122.4, 120.9, 119.0,
56.3, 56.0, 55.6, 44.3, 41.0, 37.5, 37.2. HRMS (ESI) m/z calcd for
C34H36N5O4 ([M + H]+) 578.2762; found 578.2759.
2. The synthesis of LTP
1, 2 and 3 were synthesized according to literature proce-
dures.16
(S)-(9H-Fluoren-9-yl)methyl (1-((2-(2-(dimethylamino)ethyl)-
1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamate (4). A suspension of 2 (2.55
g, 6.3 mmol) and 3 (0.60 g, 2.1 mmol) in AcOH (50 mL) was
refluxed for 4 h. The mixture was cooled, concentrated to 5 mL
and 80 mL AcOEt was added to the solution. The precipitate
was filtered and washed with 20 mL AcOEt to gain a deep
yellow powder. The deep yellow powder was recrystallized with
MeOH to produce a khaki power, 0.50 g, yield = 34.7%, mp:
139.0–141.5 uC. 1H NMR (600 MHz, DMSO) d 8.52 (t, J = 6.0 Hz,
2H), 8.36 (d, J = 6.0 Hz, 1H), 8.18 (d, J = 6.0 Hz, 1H), 8.03 (d, J =
12.0 Hz, 1H), 7.89 (d, J = 6.0 Hz, 2H), 7.83 (t, J = 6.0 Hz, 1H),
7.70 (t, J = 6.0 Hz, 2H), 7.39–7.43 (m, 4H), 7.23 (m, 5H), 7.24 (d,
J = 12.0 Hz, 1H), 4.75 (dd, J = 12.0, 6.0 Hz, 1H), 4.27 (d, J = 6.0
Hz, 2H), 4.24–4.10 (m, 3H), 3.17 (dd, J = 12.0, 6.0 Hz, 1H), 3.03
(dd, J = 12.0, 6.0 Hz, 1H), 2.22 (s, 6H), 1.91 (s, 2H).
(S)-2-Amino-N-(2-(2-(dimethylamino)ethyl)-1,3-dioxo-2,3-
dihydro-1H-benzo[de]isoquinolin-6-yl)-3-phenylpropanamide
(5). A mixture of 4 (0.30 g, 0.43 mmol) and 3 mL 20%
piperidine–DMF was stirred at room temperature for 30 min. 6
mL ether was added to the reaction solution to produce a
white power. The mother solution was added to 20 mL
n-hexane and placed at 10 uC for 2 h. The obtained precipitate
was filtered and washed with 15 mL AcOEt to give a yellow
powder, 0.14 g, yield = 75.7%. The crude powder was used in
the next step without further purification.
tert-Butyl ((R)-1-(((S)-1-((2-(2-(dimethylamino)ethyl)-1,3-
dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)amino)-1-oxo-
3-phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)carba-
mate (6). To the solution of Boc–D-Phe (0.12 g, 0.45 mmol) in
CH2Cl2 (50 mL), HOBt (0.061 g, 0.45 mmol) and EDCI (0.090 g,
0.45 mmol) were added at 0 uC. The mixture was stirred at 0 uC
for 15 min. 5 (0.13 g, 0.3 mmol) was added to the reaction
solution. After reacting at room temperature for 6 h, the
solution was washed with saturated NaHCO3 (20 mL, 4 times)
and brine (20 mL, once), and dried over anhydrous MgSO4 for
24 h. The solution was concentrated under reduced pressure
and recrystallized with MeOH–AcOEt to yield a yellowish
powder, 0.08 g, yield = 39.3%, mp: 148.1–150.0 uC. 1H NMR
(300 MHz, DMSO) d 8.67 (d, J = 8.1 Hz, 1H), 8.52 (t, J = 8.4 Hz,
2H), 8.43 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 7.85 (t, J =
8.4 Hz, 1H), 7.15–7.37 (m, 11H), 6.78 (d, J = 8.4 Hz, 1H), 4.99–
5.07 (m, 1H), 4.20–4.25 (m, 1H), 4.16 (t, J = 6.9 Hz, 2H), 3.20
(dd, J = 13.5, 5.4 Hz, 1H), 2.98 (dd, J = 13.8, 9.3 Hz, 1H), 2.71
(dd, J = 12.9, 3.6 Hz, 1H), 2.49–2.59 (m, 3H), 2.20 (s, 6H), 1.19
(s, 9H).
3. Optical property assessment
Absorption and emission spectra were primarily measured in
Britton–Robinson buffer solution (pH = 7.4). As the pH in
lysosomes is 4.5–5.0, the quantum yields of LTP, 4-NH2
naphthalimide and LT Red in Britton–Robinson buffer
solution (pH = 4.5) were obtained using fluorescein (W = 0.92
in 0.1 M NaOH) as the standard. The pH response of LTP was
tested in Britton–Robinson buffer solution with various pH
values (pH = 3.1–10.1, 7 mM), and the pKa was calculated based
on the literature.14
4. Cell culture and colocalization study of living cells
ES-2 and PC-3 cells were grown in RPMI-1640 medium
supplemented with 10% (v/v) fetal bovine serum in an
atmosphere of 5% CO2 and 95% air at 37 uC. Cells were
adjusted to the density of 80 000 per cm2, incubated for 32 h,
and then washed with cell culture medium twice. For the LTP
and LT Red co-stain, solutions of LTP (7 mM) and LT Red (70
nM) in cell culture medium were added to pre-washed cells
and incubated at 37 uC for 1 h. For the LTP and GT Red co-
stain, a GT Red work solution (prepared as per the protocol
provided by the Beyotime Institute of Biotechnology) was
added to the pre-washed cells and incubated at 4 uC for 30
min. Subsequently, the GT Red work solution was recovered
and the cells were washed three times with pre-cooled culture
medium. The solution of LTP (7 mM) in cell culture medium
was added and incubated at 37 uC for 30 min. For the LTP and
MT Green co-stain, solutions of LTP (7 mM) and MT Green (200
nM) in cell culture medium were added to the pre-washed cells
and incubated at 37 uC for 30 min. Then the samples were
observed using confocal microscopy (Zeiss LSM 700). The
colocalization coefficient and signal to noise ratio were
calculated using ImageJ software.
5. Co-staining the fixed cells with LTP and LT Red
ES-2 cells were grown in RPMI-1640 medium supplemented
with 10% (v/v) fetal bovine serum in an atmosphere of 5% CO2
and 95% air at 37 uC. Cells were adjusted to the density of
80 000 per cm2, incubated for 32 h, and then washed with cell
culture medium twice. A 3.7% formaldehyde solution was
added and incubated at 37 uC for 15 min. The cells were
washed with cell culture medium twice and solutions of LTP (7
mM) and LT Red (70 nM) in cell culture medium were added,
and incubated at 37 uC for 1 h. The samples were then imaged
(R)-2-Amino-N-((S)-1-((2-(2-(dimethylamino)ethyl)-1,3-dioxo-
2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)amino)-1-oxo-3-phe-
nylpropan-2-yl)-3-phenylpropanamide (LTP).
6
(0.050 g,
0.07mmol) in a 5 mL HClgas saturated AcOEt solution was
stirred at room temperature for 5 h and the precipitate was
filtered and washed with AcOEt (5 mL, twice). The yield
powder was dissolved in an AcOEt and saturated NaHCO3
solution (1 : 1, v/v, 40 mL). The AcOEt layer was collected and
washed with brine (10 mL, once), dried over MgSO4 and
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