A pH-resistant Zn(II) sensor derived from 4-aminonaphthalimide: Design, synthesis and intracellular applications

Article abstract of DOI:10.1039/b500766f

The development and intracellular applications of a water-soluble Zn(II) sensor (WZS) are described. 4-Aminonaphthalimide is used as the fluorophore of WZS for its high photostability, long excitation and emission wavelength, large Stokes' shift and inert response to pH. N,N-Bis(2-pyridylmethyl)ethylenediamine (BPEN), used as a receptor for Zn(II), is attached to the fluorophore through a rigid benzene framework on the imide moiety. Spectroscopic studies indicate that this fluorophore-receptor linking strategy could be used for the development of effective photoinduced electron transfer (PET) sensors. WZS is pH-insensitive and shows high selectivity for Zn(II) against other biologically relevant metal ions. Cellular applications revealed that WZS could be used for the imaging of intracellular Zn(II). The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b500766f

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www.rsc.org/materials | Journal of Materials Chemistry
A pH-resistant Zn(II) sensor derived from 4-aminonaphthalimide: design,
synthesis and intracellular applications{
Jiaobing Wang,a Yi Xiao,a Zhichao Zhang,a Xuhong Qian,*ab Yuanyuan Yanga and Qin Xua
Received 17th January 2005, Accepted 3rd March 2005
First published as an Advance Article on the web 14th March 2005
DOI: 10.1039/b500766f
The development and intracellular applications of a water-soluble Zn(II) sensor (WZS) are
described. 4-Aminonaphthalimide is used as the fluorophore of WZS for its high photostability,
long excitation and emission wavelength, large Stokes’ shift and inert response to pH. N,N-Bis(2-
pyridylmethyl)ethylenediamine (BPEN), used as a receptor for Zn(II), is attached to the
fluorophore through a rigid benzene framework on the imide moiety. Spectroscopic studies
indicate that this fluorophore–receptor linking strategy could be used for the development of
effective photoinduced electron transfer (PET) sensors. WZS is pH-insensitive and shows high
selectivity for Zn(II) against other biologically relevant metal ions. Cellular applications revealed
that WZS could be used for the imaging of intracellular Zn(II).
large Stokes’ shift, high photostability, simple structure and
Introduction
easy approaches to modification. These are all favorable pro-
Zn(II) is one of the most abundant divalent metal ions in living
perties for the design of a fluorescent chemosensor. To the best
organisms (2.3 g Zn for an average person),1 and it is an
of our knowledge, practically useful fluorescent Zn(II) sensors
essential component of many protein scaffolds (e.g., carbonic
based on this fluorophore have never been reported. We report
anhydrase and zinc finger protein).2 Zn(II) plays critical roles
here a new, pH-resistant cell-permeable zinc chemosensor
in gene transcription and metalloenzyme function.3 In addi-
that combines the advantages of the fluorophore 4-amino-
tion, the neurobiology of Zn(II) has become a subject of
naphthalimide and the widely used Zn(II) receptor BPEN.
increasing attention.4 Development of fluorescent Zn(II)
sensors would help to clarify the cellular functions of Zn(II)
Experimental
in vivo, and thus has become the focus of numerous research
efforts.5 Currently, TSQ (6-methoxy-8-(p-toluenesulfonamide)-
General considerations
quinoline) and its derivatives are the most widely used
All the solvents were of analytical grade. 1H-NMR spectra
fluorescent Zn(II) sensors. One of these derivatives, Zinquin,
were measured on a Bruker AV-400 spectrometer with
has been used to detect intracellular Zn(II). However, these
sensors require potentially damaging UV excitation.6,7
chemical shifts reported in ppm (in CDCl3, TMS as internal
standard). Mass spectra were measured on a HP 1100 LC-MS
Improvements are made by using fluorescein, one of the
spectrometer. Melting points were determined by an X-6
most popular fluorophore candidates for the development
micro-melting point apparatus and were uncorrected. All pH
of chemosensors, as fluorophore (e.g., ZnAF-1, ZnAF-2,
ZnAF-1F, ZnAF-2F, Zinpyr-1, Zinpy-r-2, Fluozin-3).7 These
measurements were made with a Sartorius basic pH-Meter PB-
20. Fluorescence spectra were determined by using a Hitachi
Zn(II)-selective chemosensors based on the PET mechanism
F-4500. Absorption spectra were determined on a PGENERAL
are successfully used for the imaging of intracellular Zn(II).
TU-1901 UV-VIS Spectrophotometer. Cell studies were
However, fluorescein itself is pH sensitive: its fluorescence
performed using a Nikon TE 2000 fluorescent microscope.
is significantly quenched due to the protonation of the
phenolic hydroxyl at lower pH values, and no fluorescence is
observed when the pH is below 4.8 Nagano and coworkers
recently reported a novel Zn(II)-selective luminescent lantha-
nide chemosensor [Eu-7] and its biological applications.9
[Eu-7] shows inert pH response, and exhibits high signal-to-
noise ratio by virtue of time-resolved imaging. However, the
cellular applications need to perform microinjection. We and
other prefer to use 4-aminonaphthalimide as fluorophore and
had already developed a series of chemosensors for different
analytes.10–12 This fluorophore absorbs and emits in the visible
wavelength range, it has the advantaged of inert pH response,
Synthesis and characterization of WZS
N-(4-Nitrophenyl)ethane-1,2-diamine (2)13. 1-Chloro-4-nitro-
benzene (1, 3.14 g, 20 mmol) and 10 ml ethane-1,2-diamine
were combined in 30 ml acetonitrile, and CuI (0.38 g, 2 mmol)
was added. The reaction mixture was stirred for 6 h under
reflux, then cooled down and poured into cold saturated brine.
The precipitation was filtered and dried to afford 2.3 g
(12.7 mmol, 63%) of yellow solid 2, which is used without
further purification. Mp 150–152 uC. MS (API-ES) calcd for
([M + H])+, 182; found, 182.
{ Electronic supplementary information (ESI) available: 1H NMR
spectra. See http://www.rsc.org/suppdata/jm/b5/b500766f/
*xhqian@ecust.edu.cn
N9-(4-Nitrophenyl)-N,N-bis(pyridin-2-ylmethyl)ethane-1,2-
diamine (3). To a solution of 0.2 g (1.1 mmol) of 2, 0.51 g
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(3.1 mmol) of 2-(chloromethyl)pyridine hydrochloride and
0.08 g (0.5 mmol) KI in 10 ml acetonitrile was added 6 ml
N,N-diisopropylethylamine. The mixture was stirred for 3 h
under reflux, then diluted with 50 ml saturated brine and
extracted with 80 ml dichloromethane three times. The organic
phase was dried over sodium sulfate and evaporated to
dryness. The residue was chromatographed on silica gel and
eluted with dichloromethane–methanol 100 : 5 (v/v) to afford
0.33 g (0.9 mmol, 82%) of yellow oil 3. 1H (400 MHz, CDCl3):
8.59 (d, 2H, J 5 4.4 Hz), 8.07 (d, 2H, J 5 9.2 Hz), 7.63 (t, 2H,
J 5 7.6 Hz), 7.35 (d, 2H, J 5 7.6 Hz), 7.19 (t, 2H, J 5 6.4 Hz),
6.88 (s, 1H), 6.51 (d, 2H, J 5 9.2 Hz), 3.94 (s, 4H), 3.22 (t, 2H,
J 5 5.6 Hz), 2.93 (t, 2H, J 5 5.6 Hz). MS (API-ES) calcd for
([M + H])+, 364; found, 364.
J 5 4.8 Hz), 8.48 (d, 1H, J 5 8.4 Hz), 8.18 (d, 1H, J 5 8.8 Hz),
7.65 (m, 3H), 7.44 (d, 2H, J 5 8.0 Hz), 7.18 (t, 2H, J 5 6.0 Hz),
7.05 (d, 2H, J 5 8.4 Hz), 6.73 (d, 1H, J 5 8.4 Hz), 6.67 (d, 2H,
J 5 8.4 Hz), 5.83 (br s,1H), 3.96 (s, 4H), 3.92 (t, 2H, J 5 5.2 Hz),
3.85 (t, 2H, J 5 4.8 Hz), 3.71 (t, 2H, J 5 4.8 Hz), 3.61 (m, 2H),
3.22 (br s, 2H), 2.95 (br s, 2H). Mp 97.8–98.3 uC. IR (KBr):
3410, 2917, 1688, 1646, 1585, 1367 cm21. HRMS (EI): [M+]
calcd for C36H36N6O4, 616.2798; found, 616.2762.
Results and discussion
Design and synthesis of WZS
The strategy for the development of WZS is as follows: (1) the
fluorophore 4-aminonaphthalimide was selected because of its
long emission wavelength, large Stokes’ shift and insensitivity
to pH; (2) N,N-bis(2-pyridylmethyl)ethylenediamine was
selected for its known affinity to Zn(II) and high potential
of cell permeance; (3) the receptor was attached to the
fluorophore through a benzene on the imide moiety using
the virtually decoupled fluorophore–receptor linking strat-
egy,14,15 the lone pair electron of the aniline nitrogen quenches
the fluorescence of the excited fluorophore 4-aminonaphtha-
limide by the PET mechanism, but when the electron-donating
aniline nitrogen is complexed with Zn(II) and the PET is
retarded, fluorescence with high quantum yield is reproduced;
(4) incorporating 2-(2-aminoethoxy)ethanol to WZS to endow
WZS with better water solubility. The synthesis of WZS is very
convenient and efficient (Scheme 1). It began with 1-chloro-4-
nitrobenzene 1. Condensation of 1 with ethane-1,2-diamine
afforded N-(2-aminoethyl)-4-nitrobenzamine 2; this was
followed by alkylation with 2-(chloromethyl)pyridine, giving
di(2-picolyl)amine derivative 3; 3 was readily reduced by
SnCl2?2H2O, affording 4; 4 was then used to react with 4-nitro-
1,8-naphthalic anhydride 5, giving 6 in a yield of 64%; finally,
stirring 6 with 2-(2-aminoethoxy)ethanol in DMF at room
temperature yielded the desired product WZS as a yellow solid
in a yield of 85%.
N-[2-(Bis{pyridin-2-ylmethyl}amino)ethyl]benzene-1,4-dia-
mine (4). To a solution of 10 ml acetonitrile and 8 ml absolute
ethanol was added 0.23 g (0.63 mmol) of 3 and 1.56 g
(6.8 mmol) of SnCl2?2H2O. The mixture was stirred for 3 h
under reflux, then neutralized with 50 ml saturated sodium
carbonate and extracted with 80 ml dichloromethane three
times. The organic phase was dried over sodium sulfate and
evaporated to dryness. The residue was chromatographed on
silica gel and eluted with dichloromethane–methanol 100 : 15
(v/v) to afford 0.19 g (0.57 mmol, 90%) of light brown oil 4. 1H
(400 MHz, CDCl3): 8.53 (d, 2H, J 5 4.8 Hz), 7.62 (t, 2H,
J 5 7.6 Hz), 7.42 (d, 2H, J 5 7.6 Hz), 7.13 (d, 2H, J 5 6.0 Hz),
6.58 (d, 2H, J 5 8.4 Hz), 6.52 (d, 2H, J 5 8.0 Hz), 3.88 (s, 4H),
3.71 (s, 2H), 3.14 (t, 2H, J 5 5.6 Hz), 2.88 (t, 2H, J 5 5.6 Hz).
MS (API-ES) calcd for ([M + H])+, 334; found, 334.
2-{4-[2-(Bis{pyridin-2-ylmethyl}amino)ethylamino]phenyl}6-
nitrobenzo[de]isoquinoline-1,3-dione (6). To a solution of 8 ml
acetic acid and 8 ml absolute ethanol was added 0.15 g
(0.45 mmol) of 4 and 0.15 g (0.61 mmol) of 4-nitro-1,8-
naphthalic anhydride 5. The mixture was stirred for 7 h under
reflux in the protection of N2, then diluted with 100 ml
saturated sodium carbonate and extracted with 80 ml
dichloromethane three times. The organic phase was dried
over sodium sulfate and evaporated to dryness. The residue
was chromatographed on silica gel and eluted with dichloro-
methane–methanol 100 : 10 (v/v) to afford 0.16 g (0.27 mmol,
64%) of brown solid 6. 1H (400 MHz, CDCl3): 8.86 (d, 1H,
J 5 8.4 Hz), 8.76 (d, 1H, J 5 7.2 Hz), 8.71 (d, 1H, J 5 8.4 Hz),
7.62 (t, 2H, J 5 7.6 Hz), 7.42 (d, 2H, J 5 7.6 Hz), 7.13 (d, 2H,
J 5 6.0 Hz), 6.58 (d, 2H, J 5 8.4 Hz), 8.59 (d, 2H, J 5 4.4 Hz),
8.41 (d, 1H, J 5 8.0 Hz), 8.00 (t, 1H, J 5 7.2 Hz), 7.67 (t, 2H,
J 5 7.6 Hz), 7.45 (d, 2H, J 5 7.6 Hz), 7.20 (t, 2H, J 5 5.6 Hz),
7.05 (d, 2H, J 5 8.4 Hz), 6.70 (d, 2H, J 5 4.8 Hz), 3.93 (s, 4H),
3.22 (t, 2H, J 5 5.6 Hz), 2.92 (t, 2H, J 5 5.6 Hz). Mp158.2–
159.0 uC. MS (APCI) calcd for ([M + H])+, 559; found, 559.
WZS. To a solution of 4 ml DMF was added 0.1 g
(0.18 mmol) of 6 and 0.19 g (1.8 mmol) of 2-(2-aminoethoxy)-
ethanol. The solution was stirred for 12 h at room tempera-
ture. The mixture was directly chromatographed on silica gel
and eluted with dichloromethane–methanol 100 : 7 (v/v) to
afford 94 mg (0.152 mmol, 85%) of yellow solid WZS. 1H
(400 MHz, CDCl3): 8.60 (d, 1H, J 5 7.2 Hz), 8.56 (d, 2H,
Scheme 1 Key: (a) ethane-1,2-diamine, acetonitrile; (b) 2-(chloro-
methyl)pyridine hydrochloride, N,N-diisopropylethylamine, acetoni-
trile; (c) SnCl2, acetonitrile–ethanol; (d) 4-nitro-1,8-naphthalic
anhydride 5, acetic acid–ethanol, N2; (e) 2-(2-aminoethoxy)ethanol,
DMF.
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Fluorescence properties of WZS
Spectroscopic studies of WZS were carried out in buffered
water solution (ethanol–water, v/v, 1 : 9). The pKa9 of WZS
was determined to be about 1.8 (Fig. 1, filled triangles). The
fluorescence intensity decreases steadily at pH above 1.2
(WF 5 0.15),16 and reaches almost a constant minimal value at
pH 3.6 (WF 5 0.03). Neither excitation nor emission wave-
length shift was observed. The concomitant change in absorp-
tion spectra of WZS is negligible (labs 5 449 nm, e 5
17100 M21 cm21). Therefore, we attribute these fluorescence
intensity changes to a PET effect, where electron transfer
from the aniline nitrogen quenches the exited state of the
4-aminonaphthalimide fluorophore, just like what happened
in the amino-substituted derivatives of fluorescein.15 Upon
addition of Zn(II) to the water solution, the quantum yield of
WZS increases significantly to 0.19. A ca. 6-fold increase in
integrated emission from 475 to 700 nm is observed (Fig. 2).
Metal-binding titrations showed that WZS forms a 1 : 1
complex with Zn(II) in aqueous solution, which is responsible
for the fluorescence enhancement. The apparent dissociation
constant (Kd 5 0.62 ¡ 0.1 nM at pH 7.0) was determined
using the literature method.17
Fig. 2 Emission spectra (excitation at 451 nm) of WZS (5 mM) in
water solution (ethanol–water 1 : 9, v/v, pH 5 7.0, 0.1 M tris-HCl,
23 uC) in the presence of different concentrations of Zn(II). The arrow
indicates the increase of Zn(II) from 0 to 8 mM. Inset: integrated
fluorescence intensity from 475 to 700 nm as a function of Zn(II)
concentration.
To explore further the utility of WZS as a pH-resistant
fluorescent Zn(II) sensor, the fluorescence intensities of the
Zn(II)–WZS complex in aqueous solution at pH values ranging
from 1.5 to 8.8 were measured (Fig. 1, empty triangles). As
expected, WZS–Zn(II) complex is not subject to interference
induced by pH changes and exhibits constant fluorescence
enhancement at pH values above 3.6.
Metal ion selectivity was examined at pH 7.0 in a buffered
tris-HCl solution (Fig. 3). Using Na+, K+, Mg2+ and Ca2+, no
changes were observed in the fluorescence emission spectra,
indicating that the receptor was not coordinating to these ions.
Pb2+, Ag+, Ni2+, Mn2+ and Fe3+ induced a slight effect on the
fluorescence of WZS, and Cu2+ quenched the fluorescence.
Like TPEN (N,N,N9,N9-tetra(2-picolyl)ethylenediamine), WZS
Fig. 3 Fluorescence intensity change profile of WZS (5 mM in 0.1 M
tris-HCl solution at pH 7.0, ethanol–water 1 : 9, v/v, 23 uC) in the
presence of various cations: 0, control; 1, Zn2+; 2, Na+; 3, K+; 4, Ag+; 5,
Ni2+; 6, Pb2+; 7, Cu2+; 8, Ca2+; 9, Fe3+; 10, Mg2+; 11, Mn2+; 12, Cd2+
;
13, Co2+. The bars represent integrated emission of WZS from 475 to
700 nm. 2 mM Na+, K+, Ca2+, and Mg2+ were added to 5 mM WZS.
25 mM other cations were added to 5 mM WZS.
probably forms complexes with these metal cations, but the
fluorescence is weakened because of an electron or energy
transfer between the metal cation and the fluorophore, which
is known as fluorescence quenching. However, Cd2+ shifted the
spectrum in the same way as Zn2+, this behavior is similar to
that observed for other Zn2+ chemosensors.5
Biological applications of WZS
Fig. 1 Dependence of the fluorescence intensity of WZS (solid
triangles) and WZS–Zn(II) complex (empty triangles) on pH in water
solution ([WZS] 5 5 mM, [Zn(II)] 5 5 mM, ethanol–water 1 : 9, v/v,
23 uC.). pH above 5.2 was buffered with 0.1 M tris-HCl, pH below
5.2 was adjusted by 75% HClO4. lex 5 451 nm. The emission was
integrated from 475 to 700 nm.
Biological applications of WZS were in cyto evaluated (we
examined the applications of WZS to cultured cells).18
Cultured HeLa cells were incubated with 10 mM WZS for
5 min, which was a sufficient time for intracellular accumula-
tion of WZS judging from its weak self-fluorescence inside a
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Jiaobing Wang,a Yi Xiao,a Zhichao Zhang,a Xuhong Qian,*ab
Yuanyuan Yanga and Qin Xua
aState Key Laboratory of Fine Chemicals, Dalian University of
Technology, PO Box 40, Zhongshan Road 158, Dalian 116012, China
bShanghai Key Laboratory of Chemical Biology, East China University
of Science and Technology, Shanghai 200237, China.
E-mail: xhqian@ecust.edu.cn; Fax: 86-411-3673488;
Tel: 86-411-3687466
Fig. 4 Fluorescence images of HeLa cells loaded with WZS (left). The
cells were incubated with 10 mM WZS for 5 min, 37 uC, under 5% CO2.
Then the cells were washed with phosphate buffered saline (PBS,
pH 5 7.4) 3 times. Fluorescence images were taken with a fluorescent
microscope (Nikon TE 2000). The excited light is WB 450–480 nm.
Fluorescence image of WZS stained cells loaded with 50 mM Zn(II) for
30 min (middle). Fluorescence image of the cells after treatment with
50 mM TPEN (right).
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We have developed a water soluble fluorescent Zn(II)-selective
sensor WZS, which uses 4-aminonaphthalimide as fluoro-
phore. BPEN, as a Zn(II) receptor, is attached to the
fluorophore through
a virtually decoupled fluorophore–
receptor linking strategy. This results in an effective PET
system. Long-wavelength excitation and emission, large
Stokes’ shift, pH insensitivity combined with cell permeance
are distinct advantages of WZS compared with some early
reported Zn(II) sensors.5 WZS has appropriate sensitivity
and may be useful for studies on the biological functions
of Zn(II).
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We acknowledge the National Key Project for Basic Research
(2003CB114400) and the National Natural Science
Foundation of China for partial support of this work.
This journal is ß The Royal Society of Chemistry 2005
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