4-Amino-1,8-naphthalimide-based fluorescent sensor with high selectivity and sensitivity for Zn2 + imaging in living cells

Article abstract of DOI:10.1016/j.inoche.2014.02.035

A new 4-amino-1,8-naphthalimide-based fluorescent sensor with iminodiacetic acid as receptor, was synthesized and characterized. Under physiological pH conditions, it demonstrates high selectivity and sensitivity for sensing Zn ^{2 +} with about 5

Full text of DOI:10.1016/j.inoche.2014.02.035

Inorganic Chemistry Communications 43 (2014) 173–178
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
4
-Amino-1,8-naphthalimide-based fluorescent sensor with high
2
+
selectivity and sensitivity for Zn imaging in living cells
a
a
a
b
c
a,
Da-Ying Liu , Jing Qi , Xiao-Yan Liu , Hua-Rui He , Jia-Tong Chen , Guang-Ming Yang ⁎
a
Department of Chemistry, Nankai University, Tianjin, China
Heowns Biochem Technologies LLC, Tianjin, China
Department of Life Sciences, Nankai University, Tianjin, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A new 4-amino-1,8-naphthalimide-based fluorescent sensor with iminodiacetic acid as receptor, was synthe-
Received 19 December 2013
Accepted 24 February 2014
Available online 28 February 2014
sized and characterized. Under physiological pH conditions, it demonstrates high selectivity and sensitivity for
sensing Zn with about 50-fold enhancement in fluorescence intensity. The fluorescent sensor exhibited a char-
2+
acteristic emission band of 4-amino-1,8-naphthalimide with a green color centered at ~550 nm and was success-
fully applied to image Zn2+ in living cells. Upon sensing of Zn the fluorescence emission spectrum is “switched
on” demonstrating the suppression of PET from the receptor to the fluorophore.
2+
Keywords:
Fluorescent sensor
Naphthalimide
Iminodiacetic acid
Zinc
©
2014 Elsevier B.V. All rights reserved.
Zinc plays an important role in many biological and environmental
processes. It is the second most abundant transition metal ions found
in physiology, where it has multiple roles in both extra- and intra-
cellular functions [1]. It is an essential element needed by human body
and is commonly found in nutritional supplements. It is believed that
disorder of zinc homeostasis is implicated in a number of diseases,
such as Alzheimer's disease, cerebral ischemia, and epilepsy [2]. Howev-
er, taking too much zinc into the body can affect your health. Therefore,
there is a great need for methods of detecting and monitoring zinc levels
in medicine and biology as well as in environment. Currently, there is
great interest in the development of fluorescent sensors for quantifying
and exploring the role of Zn2 in various aspects because of their sim-
plicity, high sensitivity, excellent selectivity and real-time detection
Herein, we report new, simple and practical 4-amino-1,8-
naphthalimide-PET-based fluorescent sensors with iminodiacetic acid
as a receptor, which is able to sense Zn2 with high selectivity and sen-
sitivity under physiological pH conditions. And it was successfully ap-
plied to image Zn2 in living cells.
+
+
Photo-induced electron transfer (PET) is an electron transfer which
occurs when certain photoactive materials interact with light. The gen-
eral design of a PET-type fluoroionophore is the “fluorophore–spacer–
receptor (ionophore)” format. A fluorescent moiety (fluorophore) is
covalently linked to an ion receptor by means of a non-π-electron-
conjugating spacer group, e.g. arkyl group with one to four carbons.
Typically, the ionophore will contain a tertiary amine; the electrons
of which can ligate the cation. In the absence of a bound cation,
the HOMO (highest occupied molecular orbital) of the unbound recep-
tor has a higher energy than the half-filled HOMO of the excited
fluorophore. This energy difference drives rapid electron transfer from
the receptor to the excited-state fluorophore, thus the fluorescence is
quenched, or “switched off”. However, when the ionophore is bound
to a cation, the energy level of the receptor's electron pair is lower
than that of the HOMO of the excited fluorophore. As a result, the iono-
phore is stabilized energetically, the electron transfer is not favored, and
thus, fluorescence is “switched on” [6].
+
[3]. However, improvements are needed to overcome several limita-
tions when they were applied to detect zinc in biological samples.
First, most of reported sensors need to be excited by UV light, which
can cause damage to living cells [4]. Second, some of reported sensors
need to be measured in organic solvent or mixed organic solvent [5].
Third, a few reported sensors have small Stokes shifts. Furthermore,
these sensors often involve lengthy and cumbersome synthesis [3]. So
far, there is no 4-amino-1,8-naphthalimide-based fluorescent sensor
available to be applied in living cells.
Therefore, we chose to use 4-amino-1,8-naphthalimide as the
fluorophore reporter in designing Sensor Zn, as it absorbs in the visible
region (λ ~ 470 nm), emits in the green (λ ~ 550 nm), with Stokes shifts
of ca. 80 nm, and possesses high fluorescence quantum yield and excel-
lent fluorescence enhancement based on photo-induced electron trans-
fer (PET) [6,7], as well as being photo-stable, in comparison with those
⁎
Corresponding author.
E-mail address: yanggm@nankai.edu.cn (G.-M. Yang).
http://dx.doi.org/10.1016/j.inoche.2014.02.035
387-7003/© 2014 Elsevier B.V. All rights reserved.
1

174
D.-Y. Liu et al. / Inorganic Chemistry Communications 43 (2014) 173–178
conventional fluorophores such as fluorescein, rhodamine, coumarins
and BODIPY, which had only survived for several weeks, stored in the
pH 7.4 HEPES at 31 °C [8] and had importantly, low sensitivity to pH be-
cause of the absence of ionizable functional groups within the physio-
logical pH range. Thanks to the powerful electron-withdrawing
property of the diimide moiety, the pKa of the amino group in the 4-
amino-1,8-naphthalimide was found to be around 2.5, much lower
than 4.5 for the unsubstituted naphthylamine. This makes the
fluorophore very insensitive to the pH near the range of physiological
pH, whose pH value varies typically between 7.34 and 7.45 [9]. Based
on above-mentioned considerations, 4-amino-1,8-naphthalimide was
selected rationally as the fluorophore.
during the wet storage at room temperature; (D) should preferentially
bind zinc with a dissociation constant (Kd) in the aqueous medium
near the desired measuring range of 1 fM in E. coli to almost 0.5 mM in
mammalian cells [10]; and (E) must have short and convenient synthe-
sis. Among the available ionophore groups, iminodiacetic acid moiety is
selected naturally as the ionophore [11].
Now, we present our design and synthesis of a new 4-amino-1,8-
naphthalimide-based fluorescent sensor with iminodiacetic acid as re-
ceptor. Scheme 1 explains the synthetic route of Sensor Zn. The detailed
procedures and characterization of the new compounds are described
in the Supporting Information.
To obtain an insight into the binding properties of Sensor Zn towards
metal ions, we investigated absorption and fluorescence changes upon
The introduction of glutamate greatly increased the solubility of the
sensor in water and helped retain the sensor inside of the cell. Before
staining the cell, the glutamate is kept as diester form, which is very
lipid soluble and diffuses readily across the lipophilic cell membranes.
Two ester groups are hydrolyzed inside of the cell by esterase, and the
resulting glutamate anions can dissolve easily in water under physiolog-
ical pH conditions, and retain inside of the stained cell for a long time.
The selection of iminodiacetic ionophore was driven by several de-
sign criteria: (A) must contain tertiary nitrogen that can act as an elec-
tron donor and will also interact with a bound zinc cation; (B) binding
properties should be insensitive to pH changes in the physiological pH
range of 7.34–7.45 so as to minimize undesirable pH interference to
+
+
2+
3+
the addition of a wide range of metal ions including Na , K , Mg
,
,
2
+
2+
2+
2+
2+
2+
2+
2+
2+
3+
Ca , Pb , Hg , Cu , Zn , Cd , Co , Mn , Fe , Fe , Cr
2
+
3+
3+
3+
Ni , La , Eu , and Er
in HEPES buffer solution (20 mM, pH =
7.4). The fluorescence changes of Sensor Zn are depicted in Fig. 1.
The addition of Zn2 to aqueous solution of Sensor Zn bearing
iminodiacetic acid as a metal chelating group caused a remarkable fluo-
rescence enhancement, about 50-fold enhancement. By contrast, minor
fluorescence enhancements were also observed upon the addition of
+
2
+
2+
Cd and Ni . No fluorescence spectral changes were observed with
2
+
2+
other metal ions. Although the addition of Cd and Ni also induced
an emission enhancement to a certain extent, Cd2 and Ni2+ which are
the highly toxic metal ions appearing in vivo are very low concentrations
+
2
+
the measurement of Zn ; (C) must possess an adequate chemostability
Scheme 1. Synthesis of Sensor Zn.

D.-Y. Liu et al. / Inorganic Chemistry Communications 43 (2014) 173–178
175
Fig. 1. (a) Fluorescence spectra of Sensor Zn (25 μM, λex = 470 nm) in the presence of var-
ious metal ions in HEPES buffer (20 mM, pH = 7.4). (b) The relative fluorescence intensity
Fig. 2. (a) Fluorescence spectra of Sensor Zn (25 μM, λex = 470 nm) in HEPES buffer
+
+
2+
2+
2+
(20 mM, pH = 7.4) in the presence of different concentration of Zn2+. (b) The correspond-
of Sensor Zn (25 μM) in the presence of various metal ions: Na , K , Mg , Ca , Pb
,
Hg , Cu , Zn , Cd , Co , Mn , Fe , Fe , Cr ,Ni and La3+, Eu3+, Er
2
+
2+
2+
2+
2+
2+
2+
3+
3+
2+
3+
ing Zn2+ titration profile of the emission at 550 nm.
in
HEPES buffer (20 mM, pH = 7.4).
[
5(c), 12]. This small interference does not affect the application of this
sensor in living cell.
The fluorescence changes of Sensor Zn (25 μM) in response to in-
M
2
(−c), 675.10, Found, 674.0 (chemical structures of M
shown below, in Fig. 4).
Therefore, based the above H NMR titrations and MS data, we show
1 2
and M are
1
2
+
2+
creasing Zn
(
concentration were measured in 20 mM HEPES buffer
the proposed binding mechanism of the Sensor Zn with Zn
Fig. 6).
(see
Fig. 2). The fluorescence emission intensity of Sensor Zn gradually in-
2
+
creased and became saturated when 6.0 equiv. of Zn was added to
Sensor Zn. The dissociation constant (Kd) between Sensor Zn and
Zn
Photo-induced electron transfer (PET) can be explained more clearly
by using this real example shown in Fig. 7. In the absence of Zn2 , the
HOMO of the unbound iminodiacetic acid moiety has a higher energy
than the half-filled HOMO of the excited 4-amino-1,8-naphthalimide.
This energy difference drives rapid electron transfer from the imino-
diacetic acid moiety to the excited-state 4-amino-1,8-naphthalimide,
+
2
+
−5
was measured to be 2.4 × 10
M by fluorescence titration
curve fitting. These results clearly demonstrate that the Sensor Zn has
excellent affinity for Zn2 over other metal ions.
+
2
+
In order to further clarify the binding property of Sensor Zn–Zn in
1
solution, H NMR titrations were carried out and the results are shown
thus the fluorescence is quenched. Consequently, it shows weak fluores-
cence. However, when the iminodiacetic acid moiety is bound to Zn
in Fig. 3. Upon the addition of Zn2 (from 0.0 equiv. to 2.0 equiv.) to the
+
2+
,
DMSO-d6 solution of Sensor Zn, the peak assigned to the proton of the
the energy level of the iminodiacetic acid moiety is lower than that of
the HOMO of the excited 4-amino-1,8-naphthalimide, and the electron
transfer is not energetically favored. The fluorescence is “switched on”.
To further demonstrate the practical biological application of Sensor
Zn, fluorescence imaging experiments were carried out in living cells.
Firstly, we had incorporated the MTT method to test the cytotoxicity
of Sensor Zn, and we found that Sensor Zn doesn't have cytotoxicity
under the concentration of 40 μM. As shown in Fig. 8, after being
H
a
moiety was gradually broadened and gotten lower, shifted downfield
from δ = 3.909 to 7.001 ppm in the end. And moreover, the peak
assigned to the proton of the H moiety was also shifted downfield
b
from δ = 6.361 to 7.001 ppm.
2
+
To further illustrate the binding property of Sensor Zn–Zn in solu-
tion, MS of Sensor Zn–Zn2 was carried out and the results are exhibit-
+
ed in Fig. 5. MS (+ESI): Calc. for M (−c), 577.17, Found, 576.2; Calc. for
1

176
D.-Y. Liu et al. / Inorganic Chemistry Communications 43 (2014) 173–178
Fig. 3. 1H NMR (300 MHz) spectra of Sensor Zn in DMSO-d6 with the addition of ZnCl
.
2
incubated with Sensor Zn (0.2 mM) in the growth medium (1 mL) for
h at 37 °C and washed with phosphate buffered saline to remove
Acknowledgments
8
excess Sensor Zn, HeLa cells displayed strong fluorescence under
irradiation with blue light. The bright-field transmission and fluores-
cence images revealed that the fluorescence signal resulted from the
intracellular region. The green fluorescence of the cells is similar to
that of Sensor Zn in solution, which indicates that Sensor Zn is mem-
brane permeable. These results suggest that Sensor Zn has good
membrane permeability and can be used as a sensor for detecting
This work was supported by the National Natural Science Founda-
tion of China (Nos. 20941004, 21071084 and 90922032), the MOE
(IRT-0927), and the Tianjin Key Laboratory of Metal and Molecule
Based Material Chemistry and Tianjin Natural Science Foundation (No.
11JCYBJC03500). The authors acknowledge the helpful discussions and
collaboration from co-workers within Heowns BioChem Technologies
LLC.
2
+
Zn
in living cells.
In conclusion, a new fluorescent sensor for Zn2 based on 4-amino-
,8-naphthalimide has been developed. The new fluorescent sensor
demonstrated highly selective and sensitive binding affinity towards
+
Appendix A. Supplementary data
1
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.inoche.2014.02.035.
2
+
Zn in HEPES buffer solution, and was applied successfully to image
2
+
Zn in living cells.
References
[
1] T. Gunnlaugsson, T.C. Lee, R. Parkesh, A highly selective and sensitive fluorescent
PET (photoinduced electron transfer) chemosensor for Zn(II), Org. Biomol. Chem.
1
(2003) 3265–3267.
[
2] (a) A.I. Bush, W.H. Pettingell, G. Malthaup, M. Paradis, J.P. Vonsattel, J.F. Gusella, K.
Beyreuther, C.L. Masters, R.E. Tanzi, Rapid induction of Alzheimer a beta amy-
loid formation by zinc, Science 265 (1994) 1464–1467;
(b) J.Y. Koh, S.W. Suh, B.J. Gwag, Y.Y. He, C.Y. Hsu, D.W. Choi, The role of zinc in se-
lective neuronal death after transient global cerebral ischemia, Science 272
(1996) 1013–1016;
(c) C.J. Frederickson, M.D. Hernandez, J.F. McGinty, Translocation of zinc may con-
tribute to seizure-induced death of neurons, Brain Res. 480 (1989) 317–321.
2
+
[
3] (a) Z. Xu, J. Yoon, D. Spring, Fluorescent chemosensors for Zn , Chem. Soc. Rev. 39
2010) 1996–2006;
(
(b) E.M. Nolan, S.J. Lippard, Small-molecule fluorescent sensors for investigating
zinc metalloneurochemistry, Chem. Rev. 46 (2009) 193–203.
[
[
4] X.B. Yang, B.X. Yang, J.F. Ge, Y.J. Xu, Q.F. Xu, J. Liang, J.M. Lu, Benzo [α]
phenoxazinium-based red-emitting chemosensor for zinc ions in biological media,
Org. Lett. 13 (2011) 2710–2713.
5] (a) Q.H. You, P.S. Chan, W.H. Chan, C.K. Hau Sam, W.M. Lee Albert, N.K. Mak, C.W.
Mak Thomas, N.S. Wong Ricky, A quinolinyl antipyrine based fluorescence sen-
2
+
sor for Zn and its application in bioimaging, RSC Adv. 2 (2012) 11078–11083;
b) H.X. Wang, H.X. Wu, L. Xue, Y. Shi, X.Y. Li, A naphthalimide fluorophore with ef-
ficient intramolecular PET and ICT processes: application in molecular logic, Org.
Biomol. Chem. 9 (2011) 5436–5444;
(
1 2
Fig. 4. Chemical structures of M and M .

D.-Y. Liu et al. / Inorganic Chemistry Communications 43 (2014) 173–178
177
Fig. 5. MS of Sensor Zn–Zn2+
.
Fig. 6. Proposed binding mechanism of the Sensor Zn with Zn2+
.

178
D.-Y. Liu et al. / Inorganic Chemistry Communications 43 (2014) 173–178
Fig. 7. PET mechanism of the Sensor Zn with Zn2+
.
Fig. 8. Fluorescence images and their corresponding bright-field transmission images: HeLa cells were incubated with Sensor Zn (2 μM) in a fresh serum free medium for 8 h, then, incu-
bated with TBAF (10 mM) in a fresh serum free medium for 0.5 h.
(
c) C.J. Gao, X.J. Jin, X.H. Yan, P. An, Y. Zhang, L.L. Liu, H. Tian, W.S. Liu, X.J. Yao, Y.
Tang, A small molecular fluorescent sensor for highly selectivity of zinc ion, Sen-
sors Actuators B Chem. 176 (2013) 775–781.
systems: approaches to sensing and switching devices via supramolecular
photophysics, Chem. Soc. Rev. 21 (1992) 187–195;
(f) A.J. Bryan, A.P. de Silva, A.R.D.D. Rupasinghe, K.R.A.S. Sandanayake,
Photo-induced electron transfer as a general design logic for fluorescent molec-
ular sensors for cations, Biosensors 4 (1989) 169–179.
[
6] (a) A.P. de Silva, H.Q.N. Gunaratne, T. Gunnlaugsson, A.J.M. Huxley, C.P. McCoy, J.T.
Rademacher, T.E. Rice, Signaling recognition events with fluorescent sensors
and switches, Chem. Rev. 97 (1997) 1515–1566;
[8] (a) S. Lee, J.H. Lee, T. Pradhan, C.S. Lim, B.R. Cho, S. Bhuniya, S.J. Kim, J.S. Kim, Fluo-
2
+
(b) A.P. de Silva, T.S. Moody, G.D. Wright, Fluorescent PET (Photoinduced Elec-
rescent turn-on Zn sensing in aqueous and cellular media, Sensors Actuators
B Chem. 160 (2011) 1489–1493.
tron Transfer) sensors as potent analytical tools, Analyst 134 (2009)
2
385–2393.
(b) H. He, Unpublished results.
[
7] (a) S.R. Davidson, The chemistry of excited complexes: a survey of reactions, Adv.
[9] F. Walter Boron, E.L. Boulpaep, Medical Physiology: a Cellular and Molecular Ap-
proach, Elsevier/Saunders, 2004.
Phys. Org. Chem. 19 (1983) 1–130;
(
(
b) V. Balzani (Ed.), Electron Transfer in Chemistry, Wiley-VCH, Weinheim, 2001;
c) S.C. Burdette, G.K. Walkup, B. Spingler, R.Y. Tsien, S.J. Lippard, Fluorescent sen-
sors for Zn based on a fluorescein platform: synthesis, properties and intracel-
lular distribution, J. Am. Chem. Soc. 123 (2001) 7831–7841;
[10] (a) C.J. Federickson, Neurobiology of zinc and zinc-containing neurons, Int. Rev.
Neurobiol. 31 (1989) 145–238;
2
+
(b) C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and dis-
ease, Nat. Rev. Neurosci. 6 (2005) 449–462.
(
d) K. Yoshida, T. Mori, S. Watanabe, H. Kawai, T. Nagamura, Synthesis and metal
ion-sensing properties of fluorescent PET chemosensors based on the
[11] R. Parkesh, T.C. Lee, T. Gunnlaugsson, Highly selective 4-amino-1,8-naphthalimide
based fluorescent photoinduced electron transfer (PET) chemosensors for Zn(II)
under physiological pH conditions, Org. Biomol. Chem. 5 (2007) 310–317.
[12] (a) Nickel, Regional office for Europe, 2000. (Copenhagen, Denmark);
(b) Thomas J. Lyons, David J. Eide, Transport and Storage of Metal Ions in Biology,
2006. 57.
2-phenylimidazo[5,4-α]anthraquinone chromophore, J. Chem. Soc. Perkin
Trans. 2 (1993) 393–398;
(e) R.A. Bissell, A.P. de Silva, H.Q.N. Gunaratne, M.P.L. Lynch, G.E.M. Maguire, K.R.A.S.
Sandanayake, Molecular fluorescent signalling with ‘fluor–spacer–receptor’