A naphthalimide fluorescent sensor for Zn2+ based on photo-induced electron transfer

Article abstract of DOI:10.1246/cl.2004.1392

A new Zn²⁺ fluorescent sensor NIDPA (1) which takes 1,8-naphthalimide as a reporting group and di-2-picolylamine (DPA) as a recognizing group has been synthesized via simple steps. Based on photo-induced electron transfer (PET) mechanism, NIDPA has a nearly 5-fold fluorescent enhancement under simulated physiological conditions corresponding to the binding of Zn²⁺. Apparent dissociation constant for Zn²⁺ (K_d) is in the sub-nM range, and Ca²⁺, Mg²⁺, Fe³⁺, Ni²⁺, and Cr³⁺ have little influence on fluorescence enhancement.

Full text of DOI:10.1246/cl.2004.1392

1392
Chemistry Letters Vol.33, No.10 (2004)
A Naphthalimide Fluorescent Sensor for Zn2þ Based on Photo-induced Electron Transfer
Jiangli Fan, Yunkou Wu, and Xiaojun Pengꢀ
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, P. R. China
(Received July 7, 2004; CL-040809)
A new Zn2þ fluorescent sensor NIDPA (1) which takes 1,8-
naphthalimide as a reporting group and di-2-picolylamine
(DPA) as a recognizing group has been synthesized via simple
steps. Based on photo-induced electron transfer (PET) mecha-
nism, NIDPA has a nearly 5-fold fluorescent enhancement under
simulated physiological conditions corresponding to the binding
of Zn2þ. Apparent dissociation constant for Zn2þ (Kd) is in the
sub-nM range, and Ca2þ, Mg2þ, Fe3þ, Ni2þ, and Cr3þ have little
influence on fluorescence enhancement.
N NaOH (ꢀ ¼ 0:85) as a standard. The low quantum yield of
the bound-free sensor results from PET quenching, which is
due to the electron transfer from the nitrogen atoms in DPA moi-
ety to the fluorophore. Upon addition of Zn2þ, the fluorescence
intensity was increased by nearly 5-fold, and the quantum yield
of the Zn2þ complex was increased up to 0.17. The absorption
maximum wavelength and the emission maximum wavelength
were blue shifted to 443 and 539 nm, which accounts for the co-
ordination of the amino group on naphthalene with Zn2þ
.
5
4
3
2
1
Zn2þ is one of the most important transition metal ions
found in physiology, where it has multiple roles in both extra-
and intra-cellular functions.1,2 Some of the roles have been long
known, such as in gene transcription and metallloenzyme.2,3
Other functions are just being discovered and investigated, such
as zinc’s role in synaptic neurotransmission4 and in mediation
neuronal excitotoxicity.5 Currently, there is great interest in
the development of fluorescent sensors for exploring the role
of Zn2þ in medicine and biology as well as in the environ-
ment.6–10 However, there is still scope for improvement in the
design of such sensors as they often suffer from disadvantages
such as sensitivity to Hþ, Ca2þ, and Mg2þ, short excitation
and emission wavelengths, small Stokes shifts and cumbersome
synthesis.
0
noneMg2+Ca2+ K+ Cu2+Co2+Ni2+ Ag+ Cr3+Mn2+Cd2+Zn2+
Metal cation
Figure 1. Fluorescence response of 1 mM NIDPA to various
metal cations. Bars represent the final integrated fluorescence
response (F) over the initial intergtated emission (F0). Initial
spectrum was acquired in 100 mM NaCl, 10 mM Tris, 1 mM
EDTA pH 7.4 at 25 ꢁC.
Herein, we report the design and properties of (NIDPA, 1), a
new Zn2þ selective and sensitive fluorescent PET sensor synthe-
sized in a few steps with high yields. In NIDPA, 4-aminonaph-
thalimide, with large Stockes shift and desirable spectroscopic
properties is used as fluorophore, and N,N-bis(2-pyridylmethyl)
ethylenediamine (DPA), a selective chelator for Zn2þ, as recep-
tor. The nitrogen atom of tertiary amine is both the chelating site
of Zn2þ and the electron-source for PET. The synthetic proce-
dure is shown in Scheme 1. 4 was prepared according to the sim-
ilar method previously described with 70% yield.11 NIDPA (1)
was easily synthesized via the reaction of 2-chloromethylpyri-
dine and 4 with 50% yield, and characterized by spectroscopic
data.12
The fluorescence response of NIDPA to various cations is
shown in Figure 1. As expected, other physiologically important
cations which exist at high concentration in living cells such as
Ca2þ, Mg2þ, Naþ, and Kþ did not give rise to any changes in the
fluorescence emission of NIDPA even at high concentration
(5 mM). These results are presumably due to the poor complex-
ation of alkaline metals or alkaline earth metals with the chelator
of NIDPA. Similar results were obtained for some first-row tran-
sition metal cations, their fluorescence spectra were slightly in-
fluenced by addition of Fe3þ, Ni2þ, and Cr3þ (5 mM). Cu2þ
and Co2þ quenched the fluorescence to a little extent, probably
because there is electron or energy transfer between metal cation
and fluorophore, which is known as the fluorescence quenching
mechanism.13 But these cations would have little influence in
vivo, since they are not present to a significant extent in ordinary
biological system.
CH3
CH3
N
CH3
N
O
N
O
O
O
O
O
O
O
O
NH2.CH3
NH2CH2CH2NH2
Cl
N
N
Br
Br
N
HN
NHCH2CH2NH2
2
3
4
1
N
The sensors based on PET are usually disturbed by proton in
the detection of metal cations, so the low sensitivity to the oper-
Scheme 1. Synthesis of NIDPA.
The absorption maximum wavelength of NIDPA was
ative pH is very important.14 In the presence of saturating Zn2þ
,
453 nm, emission maximum wavelength was 549 nm, and fluo-
rescence quantum yield was 0.04 under physiological conditions
(pH 7.4, I ¼ 0:1 (NaCl)) with EDTA to scavenge adventitious
metal ions, which was determined by using fluorescein in 0.1
the fluorescence intensity of NIDPA decreases along with the in-
crease of pH value, but when pH >7:0, the fluorescence intensity
is almost a constant (Figure 2). From the change of fluorescence
intensity integrated from 480 to 700 nm, a sigmoidal curve is ob-
Copyright ꢀ 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.10 (2004)
1393
served, which gives a pKa of 5.8, which corresponds to proton-
ation of the tertiary nitrogen atom of DPA. So, it is almost stable
over the physiological pH range.
nm with 5-fold fluorescence enhancement after complexation
with Zn2þ. The quantitative response range is in the sub-nM
range, and its fluorescence is not induced by other biologically
important cations such as Kþ, Naþ, Ca2þ, Fe3þ, Ni2þ, and
Mg2þ under physiological conditions.
8
7
6
5
4
3
This work was supported by ‘‘973 program’’ of the Ministry
of Science and Technology of China and National Science
Foundation of China (project 20128005, 20376010).
References and Notes
1
See special issue on Zn(II) chemistry: W. Maret, BioMetals,
14, 187 (2001).
2
3
4
J. M. Berg and Y. Shi, Science, 271, 1081 (1996).
B. L. Vallee and K. H. Falchuk, Physiol. Rev., 73, 9 (1993).
D. W. Choi and J. Y. Koh, Annu. Rev. Neurosci., 21, 347
(1998).
2
4
6
8
10
12
pH
5
6
J. H. Weiss, S. L. Sensi, and J. Y. Koh, Trends Pharmacol.
Sci., 21, 347 (2000).
Figure 2. Effect of pH on the fluorescence intensity of 1 mM
NIDPA–Zn2þ in phosphatic buffers (excitation at 453 nm) at
25 ꢁC.
a) R. B. Thompson, D. Peterson, W. Mahoney, M. Cramer,
B. P. Maliwal, S. W. S. Suh, C. Frederickson, C. Fierke,
and P. Herman, J. Neurosci. Methods, 118, 63 (2002).
b) E. Kimura and S. Aoki, BioMetals, 14, 191 (2001). c)
E. Kimura and S. Aoki, Chem. Soc. Rev., 27, 179 (1998).
a) S. C. Burdette, G. K. Walkup, B. Spingler, R. Y. Tsien,
and S. J. Lippard, J. Am. Chem. Soc., 123, 7831 (2001). b)
S. C. Burdette and S. J. Lippard, Inorg. Chem., 25, 6816
(2002). c) S. C. Burdette, C. J. Frederickson, W. Bu, and
S. J. Lippard, J. Am. Chem. Soc., 125, 1778 (2003).
a) T. Hirnao, K. Kikuchi, Y. Urano, T. Hibuchi, and T.
Nagano, Angew. Chem., Int. Ed., 39, 1052 (2000). b) T.
Hirano, K. Kikuchi, Y. Urano, and T. Nagano, J. Am. Chem.
Soc., 124, 6555 (2002). c) S. Maruyama, K. Kikuchi, T.
Hirano, Y. Urano, and T. Nagano, J. Am. Chem. Soc., 124,
10650 (2002).
8
1.0
7
7
8
0.8
6
0.6
0.4
5
0.2
4
0.0
0
5
10
15
20
[Zn2+]/ nM
3
2
1
0
500
550
600
Wavelength / nm
650
700
9
a) T. Gunnlaugsson, T. C. Lee, and R. Parkesh, Org. Biomol.
Chem., 1, 3265 (2003). b) M. D. Shults, D. A. Pearce, and
B. Imperiali, J. Am. Chem. Soc., 125, 10591 (2003). c)
S. A. de Silva, A. Zavaleta, D. E. Baron, O. Allam, E. V.
Isidor, N. Kashimura, and J. M. Percarpio, Tetrahedron Lett.,
38, 2237 (1997).
Figure 3. Fluorescence emission spectra of 1 mM NIDPA in
buffered Zn2þ solutions with free Zn2þ concentrations from 0
to 20 nM, for the final several spectra, additional ZnSO4 was
added to provide the concentration of free Zn2þ to 25 mM. Inset:
fluorescence response obtained by integrating the emission spec-
10 a) M. E. Huston, C. Engleman, and A. Cadmiation, J. Am.
Chem. Soc., 112, 7054 (1990). b) S. Aoki, S. Kaido, H.
Fujioka, and E. Kimura, Inorg. Chem., 42, 1023 (2003).
11 X. H. Qian, Z. H. Zhu, and K. C. Chen, Dyes Pigm., 11, 13
(1989).
tra between 480 and 700 nm, subtracting the baseline (0 Zn2þ
)
spectrum and normalizing to the full scale. These data were
measured in 10 mM Tris-HCl solutions (pH 7.4) containing
100 mM NaCl, 10 mM NTA, and 0–10 mM ZnSO4. The slit
width was 4 nm for both excitation and emission.
12 For 1: mp 163–164 ꢁC. 1H NMR (400 MHz, CDCl3): ꢀ 8.82
(d, 1H, J ¼ 8:4), 8.64 (d, 1H, J ¼ 7:2), 8.57 (d, 2H, J ¼ 4:8),
8.43 (d, 1H, J ¼ 8:4), 7.82 (s, 1H), 7.72 (t, 1H, J ¼ 8:0), 7.58
(t, 2H, J ¼ 8:0), 7.41 (d, 2H, J ¼ 7:6), 7.16 (t, 2H, J ¼ 5:6),
6.54 (d, 1H, J ¼ 8:4), 4.03 (s, 4H), 3.54 (s, 3H), 3.42 (s, 2H),
3.09 (s, 2H). 13C NMR (100 MHz, CDCl3): ꢀ 165.2, 164.6,
156.7, 148.8, 137.4, 134.7, 131.2, 130.9, 129.6, 128.9,
128.1, 124.6, 124.3, 123.0, 122.7, 120.9, 112.0, 59.5, 51.2,
40.7, 27.1. API–ES–MS (positive) m=z: 452 ([M + H]þ).
13 P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher, and T. E. Rice,
Chem. Rev., 97, 1515 (1997).
In order to determine the ability of NIDPA to complex with
Zn2þ, the apparent dissociation constant, Kd,, was determined
using Zn2þ and pH-buffered solutions8b (Figure 3). From the
sigmoidal curve, Kd is 0.83 nM. Hence, NIDPA can be used to
determine free Zn2þ concentrations at low levels, which is
sensitive for application in mammalian cells. Furthermore, a
Hill plot analysis revealed maximum fluorescence obtained
at 1:1 ratio, which suggested that NIDPA should form a 1:1
complex with Zn2þ
.
In summary, we have developed a simple and sensitive flu-
orescent probe NIDPA (1) for Zn2þ with N,N-bis(2-pyridylme-
thyl)ethylenediamine as acceptor for Zn2þ and 1,8-naphthal-
imide as a fluorophore. It is excited at 453 and emits at 539
14 G. Klein, D. Kaufmann, S. Schurch, and J. L. Reymond,
¨
Chem. Commun., 2001, 561.
Published on the web (Advance View) September 25, 2004; DOI 10.1246/cl.2004.1392