Fluorescent chemosensor for biological zinc ions

Article abstract of DOI:10.1080/10610278.2012.720020

A synthetic fluorescent sensor (sensor 1) is developed using boron-dipyrromethene as a fluorophore and phenoxo-bridged dipicolylamine as a receptor. Sensor 1 shows a significant enhancement of fluorescent intensity and a high selectivity for Zn²⁺ over various metal cations. The electrochemical study provides a rationale for the fluorescence turn-on of sensor 1 upon complexation with Zn²⁺ by the blocking of the photo-induced electron transfer mechanism. Sensor 1 can be used to image Zn ²⁺ in A549 cells.

Full text of DOI:10.1080/10610278.2012.720020

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Supramolecular Chemistry
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Fluorescent chemosensor for biological zinc ions
Se Won Bae a , Eunha Kim a b , Ik-Soo Shin a c , Seung Bum Park a b & Jong-In Hong a
a Department of Chemistry, Seoul National University, Seoul, 151-747, South Korea
b Department of Biophysics and Chemical Biology/BioMAX Institute, Seoul National
University, Seoul, 151-747, South Korea
c Department of Chemistry, Soongsil University, Seoul, 156-743, South Korea
Version of record first published: 20 Sep 2012.
To cite this article: Se Won Bae , Eunha Kim , Ik-Soo Shin , Seung Bum Park & Jong-In Hong (2013): Fluorescent chemosensor
for biological zinc ions, Supramolecular Chemistry, 25:1, 2-6
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Supramolecular Chemistry
Vol. 25, No. 1, January 2013, 2–6
Fluorescent chemosensor for biological zinc ions
Se Won Baea, Eunha Kima,b, Ik-Soo Shina,c, Seung Bum Parka,b* and Jong-In Honga*
b
aDepartment of Chemistry, Seoul National University, Seoul 151-747, South Korea; Department of Biophysics and
Chemical Biology/BioMAX Institute, Seoul National University, Seoul 151-747, South Korea; Department of Chemistry,
Soongsil University, Seoul 156-743, South Korea
c
(Received 21 June 2012; final version received 7 August 2012)
A synthetic fluorescent sensor (sensor 1) is developed using boron-dipyrromethene as a fluorophore and phenoxo-bridged
dipicolylamine as a receptor. Sensor 1 shows a significant enhancement of fluorescent intensity and a high selectivity for
Zn2þ over various metal cations. The electrochemical study provides a rationale for the fluorescence turn-on of sensor 1
upon complexation with Zn2þ by the blocking of the photo-induced electron transfer mechanism. Sensor 1 can be used to
image Zn2þ in A549 cells.
Keywords: fluorescent sensor; zinc ion; PeT; cell imaging
Introduction
Results and discussion
Zn2þ is an important divalent metal cation in biological
systems, which plays a critical role in DNA synthesis,
enzyme regulation, neural signal transmission and gene
expression (1–4). While most Zn2þ ions are bound to
proteins, the disruption of mobile Zn2þ is associated with a
number of diseases such as ischaemia, epilepsy, Parkin-
son’s disease, Alzheimer’s disease and certain types of
cancer (5–8). Moreover, variation in the intracellular
mobile Zn2þ concentration is a significant factor affecting
the early apoptotic process (9–12). Quantitative analysis
of trace Zn2þ using a chemosensor comprising a selective
analytical receptor and a sensitive fluorophore has become
extremely important for environmental and biological
applications. One of the most widely applied strategies for
the development of effective fluorescent Zn2þ sensors is
the fluorescent turn-on mechanism by the regulation of
photo-induced electron transfer (PeT) (13–15). A large
number of Zn2þ sensors have been reported so far, and
significant efforts are continuing for the development of
more effective sensors to understand the biologically
crucial role of Zn2þ (reviews: 16–20, selected recent
examples: 21–25).
First, the fluorescence emission changes of sensor 1 (1 mM)
were measured as a function of Zn2þ (perchlorate salt)
concentration. Upon the addition of Zn2þ, the fluorescence
emission intensity of sensor 1 gradually increased. When
10 equiv. of Zn2þ were added to sensor 1, the emission
intensity ratio (I/I0) showed a 4.5-fold enhancement
(Figure 1).
The selectivity of sensor 1 for Zn2þ over other metal
ions was evaluated. The experimental results show that the
existence of a 10-fold excess of Liþ, Naþ, Kþ, Ca2þ
,
Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Hg2þ or Pb2þ
causes fluorescence quenching (Liþ, Ca2þ, Mn2þ, Fe2þ
,
Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Hg2þ and Pb2þ) or no
fluorescence change (Naþ and Kþ), and that only Zn2þ
leads to fluorescence enhancement (Figure 2).
To demonstrate the preferential binding ability of
sensor 1 to Zn2þ over other transition metal ions, we
prepared a 1 mM solution of sensor 1 with a 10-fold excess
of various transition metal ions. The change in
fluorescence intensity upon the addition of Zn2þ was
measured (Figure 3). In the presence of various metal ions,
the fluorescence intensity of sensor 1 decreased compared
to its initial fluorescence intensity. When 10 equiv. of Zn2þ
ions were added to the solution, the fluorescence intensity
ratio showed a ninefold enhancement. The increase in
fluorescence intensity upon treatment with Zn2þ ions is
attributed to the binding of Zn2þ to the nitrogen atoms and
a phenolate oxygen atom in sensor 1. This, in turn, blocked
the PeT, resulting in an increase in the fluorescence
intensity. The fluorescence turn-on mechanism was
rationalised by electrochemical studies (Figure 4). The
In this article, we describe the design, mechanism and
evaluation of the Zn2þ fluorescent chemosensor, sensor 1
(Scheme 1). The selectivity and the sensitivity of sensor 1
for Zn2þ were examined. The fluorescence turn-on of
sensor 1 upon the addition of Zn2þ is explained by the
blocking of PeT from the electron donor. The utility of
sensor 1 for Zn2þ sensing was demonstrated through the
imaging of intracellular Zn2þ in A549 cells using
fluorescent microscopy.
*Corresponding authors. Email: sbpark@snu.ac.kr; jihong@snu.ac.kr
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2013 Taylor & Francis
http://dx.doi.org/10.1080/10610278.2012.720020
http://www.tandfonline.com

Supramolecular Chemistry
3
(a)
(b)
F
F
F
F
B
B
N
N
N
N
CHO
CHO
Zn2+
N
N
N
N
N
N
N
N
N
N
N
N
-
O
N
N
Zn2+
N
OH
2
OH
3
OH
1
N
Zn2+
N
N
Fluorescent
Scheme 1. Synthetic procedure for sensor 1. (a) DPA, formaldehyde solution (37 wt%), cat. HCl, ethanol, reflux. (b) (i) 2,4-Dimethyl-3-
ethylpyrrole, cat. trifluoroacetic acid (TFA), 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), CH2Cl2, rt; (ii) BF3-OEt2, TEA, CH2Cl2, rt.
Figure 3. Changes in the fluorescence intensity þof seþnsorþ1
Figure 1. Changes in the fluorescence intensity of sensor 1
(1 mM) and various cations (perchlorate salts of Li , Na , K ,
(1 mM) in acetonitrile upon addition of increasing amounts of
Ca2þ, Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Agþ, Cd2þ, Hg2þ, Pb2þ
Zn2þ (excitation at 490 nm). The arrow indicates the direction of
ions, 10 mM each) upon addition of increasing amounts of Zn2þ
[Zn2þ] increase; [Zn2þ] ¼ 0, 0.2, 0.4, 0.9, 1.3, 1.8, 3.0, 4.0, 5.0,
(perchlorate salt) in acetonitrile at 258C. The arrow indicates the
7.0 and 10 mM.
direction of [Zn2þ] increase; [Zn2þ] ¼ 0, 0.2, 0.4, 0.9, 1.3, 1.8,
3.0, 4.0, 5.0, 7.0 and 10 mM.
obtained electrochemical data explain the changes in
fluorescence intensity upon the addition of Zn2þ to sensor
1. When Zn2þ binds to sensor 1, there is a decrease in the
electron density on the two nitrogen atoms of the amine
moiety. This leads to the stabilisation of the highest
occupied molecular orbital (HOMO) of sensor 1. In order
to investigate the variations in the HOMO of sensor 1, we
measured the oxidation potential of 2,6-bis((bis(pyridin-2-
ylmethyl)amino)methyl)phenol (DiDPA) (see Scheme 2
for the structure of DiDPA) before and after the addition of
Zn2þ ions. As shown in Figure 4, the first oxidation
potential of DiDPA (E1/2 ¼ 0.83 V) corresponds to the
HOMO level of the amine moiety in DiDPA, and this
potential becomes more positive (E1/2 ¼ 1.17 V) upon the
addition of Zn2þ. The incorporation of Zn2þ into the
DiDPA moiety leads to the stabilisation of the HOMO of
Figure 2. Relative fluorescence intensity profiles of sensor 1
DiDPA through a decrease in the electron density on the
(1 mM) in acetonitrile in the presence of various cations
DiDPA nitrogen atoms, and hence, the oxidation potential
(10 equiv.). Excitation wavelength was set at 490 nm and
emission was monitored at 536 nm.
becomes more positive.

4
S.W. Bae et al.
Figure 4. Cyclic voltammograms of (a) 2 mM DiDPA molecule and (b) 2 mM DiDPA after addition of 2 mM Zn2þ (with 0.2 M TBAPF6
supporting electrolyte in acetonitrile).
F
F
B
N
N
N
N
N
N
N
N
OH
BODIPY
DiDPA
Scheme 2. Chemical structures of Bodipy and DiDPA.
On the basis of the electrochemical data, the
enhancement of the fluorescence intensity can be
explained from a thermodynamics viewpoint (Scheme 3).
The electronic states of sensor 1 correspond to those of
boron-dipyrromethene (Bodipy) and DiDPA. Since these
two moieties are bonded in an orthogonal fashion, their
electronic states are independent of each other, and thus,
only intramolecular through-space electron transfer is
allowed. Therefore, in the absence of Zn2þ, an electron can
Figure 5. Fluorescence microscopy images (600 £ ) of A549
cells, incubated without (a) and with (b) 10 mM sensor 1 for
30 min. Increased fluorescence signal is observed with the
addition of 50 mM ZnCl2 and 10 mM 2-pyrithione according to
incubation time: 5 min (c), 15 min (d) and 30 min (e). TPEN
addition induces the decrease in fluorescence signal according to
incubation time: 5 min (f), 15 min (g) and 30 min (h).
Quantification of the fluorescence intensity according to time (i).
be transferred easily from the HOMO of DiDPA to the
half-filled ground state of the excited Bodipy moiety,
*
which subsequently leads to the efficient fluorescent
quenching of sensor 1. In the presence of Zn2þ, however,
the HOMO of DiDPA is stabilised because of a decrease in
the electron density on the nitrogen atoms of the DiDPA
moiety. Hence, upon the addition of Zn2þ, fluorescence
enhancement occurs through the blocking of PeT from the
HOMO of DiDPA to the half-filled ground state of the
Scheme 3. Thermodynamic explanation of fluorescent
enhancement upon Zn2þ addition to sensor 1. Energetic values
of Bodipy show a slight shift when this moiety is incorporated
into sensor 1.
excited Bodipy moiety.
*

Supramolecular Chemistry
5
The fidelity of sensor 1 for the sensing of Zn2þ in
biological systems was examined with fluorescence
microscope images obtained using A549 cells (Figure 5).
The cells were seeded on a cover-glass-bottomed Petri dish
and incubated overnight. The cells were then washed with
phosphate-buffered saline (PBS buffer) solution for 5 min
each, and sensor 1 (10 mM in PBS buffer) was treated for
30 min. Then, 2-pyrithione (10 mM) and ZnCl2 (50 mM)
were added. The fluorescence signal was measured at each
time point. After 30 min, N,N,N0,N0-tetrakis(2-pyridyl-
methyl)ethylenediamine (TPEN) (100 mM) was added to
the A549 cells without washing, and the fluorescence
signal was measured at each time point. The strong
intracellular fluorescence of sensor 1 was observed until
the well-known metal chelator, TPEN, induced the
decrease in the fluorescence signal. Figure 5(i) shows the
quantification of the fluorescence intensity according to
the time domain.
tography monitoring (silica; CH2Cl2) showed complete
consumption of the aldehyde, a solution of 2,3-dichloro-
5,6-dicyanobenzoquinone (300 mg, 1.1 mmol) in CH2Cl2
was added, and stirring was continued for 15 min. The
reaction mixture was washed with H2O, dried over
Na2SO4, filtered and evaporated. The crude compound
(387 mg, 0.5 mmol) and triethylamine (TEA, 1.3 mL,
9.38 mmol) were dissolved in 60 mL of absolute CH2Cl2
under N2 atmosphere and stirred at room temperature for
10 min. Then, BF3-OEt2 (1.3 mL, 9.37 mmol) was added,
and stirring was continued for 1 h. The reaction mixture
was washed with H2O and 2 N NaOH. The aqueous
solution was extracted with CH2Cl2. The combined
organic extracts were dried over Na2SO4, filtered and
evaporated. The crude compound was purified by
column chromatography over aluminium oxide (CH2Cl2/
MeOH, 20:1) to afford a purple powder, sensor 1 (312 mg,
yield 38%).
Conclusion
Electrochemical measurements
In summary, sensor 1 binds to Zn2þ ions in preference to
other transition metal ions. Hence, it can be used to
quantify the concentration of Zn2þ in the presence of other
transition metal ions. Furthermore, electrochemical
studies reveal that the fluorescence intensity of sensor 1
increases upon the addition of Zn2þ through blocking of
PeT from the HOMO of DiDPA to the half-filled ground
The electrochemical study was conducted using a CH
Instruments 660 Electrochemical Analyzer (CH Instru-
ments, Inc., Austin, TX, USA). In the electrochemical
study, cyclic voltammetry and differential pulse voltam-
metry were performed on the individual solutions in order
to investigate their electrochemical oxidative and reduc-
tive behaviours. All the electrochemical experiments were
referenced with respect to an Ag/Agþ reference electrode.
All potential values were calibrated against the saturated
calomel electrode (SCE) by measuring the oxidation
potential of 1 mM ferrocene (vs Ag/Agþ) as a standard
(E8(Fcþ/Fc) ¼ 0.424 V vs SCE).
state of the excited Bodipy moiety. We have also shown
*
that sensor 1 can image the intracellular Zn2þ of A549
cells. This approach is expected to be relevant to various
biomedical applications, and may lead to an improved
understanding of intracellular Zn2þ
.
Experimental details
Acknowledgement
Synthesis of sensor 1
This work was supported by the NRF grant funded by the MEST
(Grant No. 2012-0000159).
Sensor 1 was prepared according to the reported procedure
(26). Briefly, 5.9 g of 37% aqueous formaldehyde solution
was added to ethanol and then to a catalytic amount of
HCl. Then, 2,20-dipicolylamine (DPA, 11.8 mL) was added
to this solution. The reaction mixture was heated at reflux
for 1 day. Subsequently, 4 g of 4-hydroxybenzaldehyde
was added to the reaction mixture. After stirring for 2 days
at reflux, the reaction mixture was cooled to room
temperature and concentrated in vacuo to remove all
volatiles. The residue was subjected to silica column
chromatography (CH2Cl2/MeOH) to give the desired
product 2 (8.6 g, yield 48%).
References
(1) Jiang, P. Coord. Chem. Rev. 2004, 248, 205–229.
(2) Burdette, S.C.; Lippard, S.J. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 3605–3610.
(3) Pluth, M.D.; Tomat, E.; Lippard, S.J. Annu. Rev. Biochem.
2011, 80, 333–355.
(4) Hambidge, M.; Cousins, R.J.; Costello, R.B. J. Nutr. 2000,
130, 1341S–1343S.
(5) Frederickson, C.J.; Koh, J.-Y.; Bush, A.I. Nat. Rev.
Neurosci. 2005, 6, 449–462.
(6) Takeda, A.; Tamano, H. Brain Res. Rev. 2009, 62, 33–44.
(7) Sensi, S.L.; Paoletti, P.; Bush, A.I.; Sekler, I. Nat. Rev.
Neurosci. 2009, 10, 780–791.
(8) Lu, M.; Fu, D. Science 2007, 317, 1746–1748.
(9) Treves, S.; Trentini, P.L.; Ascanelli, M.; Bucci, G.;
DiVirgilio, F. Exp. Cell Res. 1994, 211, 339–343.
Compound 2 (654 mg, 1.2 mmol) and 2,4-dimethyl-3-
ethylpyrrole (0.32 mL, 2.4 mmol) were dissolved in 50 mL
of absolute CH2Cl2 under an Ar atmosphere. One drop of
trifluoroacetic acid was added, and the solution was stirred
at room temperature overnight. When thin-layer chroma-

6
S.W. Bae et al.
(10) Truong-Tran, A.Q.; Carter, J.; Ruffin, R.E.; Zalewski, P.D.
Biometals 2001, 14, 315–330.
(18) Carol, P.; Sreejith, S.; Ajayaghosh, A. Chem. Asian J. 2007,
2, 338–348.
(11) Kimura, E.; Takasawa, R.; Tanuma, S.; Aoki, S. Sci. STKE
2004, pl7, 223.
(19) Callan, J.F.; de Silva, A.P.; Magri, D.C. Tetrahedron 2005,
61, 8551–8588.
(12) Kimura, E.; Aoki, S.; Kikuta, E.; Koike, T. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 3731–3736.
(13) de Silva, A.P.; de Silva, S.A. Chem. Commun. 1986,
1709–1710.
(14) de Silva, A.P.; Gunaratne, H.Q.N.; Gunnlaugsson, T.;
Huxley, A.J.M.; McCoy, C.P.; Riademacher, J.T.; Rice,
T.E. Chem. Rev. 1997, 97, 1515–1566.
(15) Moore, E.G.; Bernhardt, P.V.; Fu¨rstenberg, A.; Riley, M.J.;
Smith, T.A.; Vauthey, E. J. Phys. Chem. A 2005, 109,
3788–3796.
(16) Huang, Z.; Lippard, S.J. Methods Enzymol. 2012, 505,
445–468.
(20) Kikuchi, K.; Komatsu, K.; Nagano, T. Curr. Opin. Chem.
Biol. 2004, 8, 182–191.
(21) Sun, F.; Zhang, G.; Zhang, D.; Xue, L.; Jiang, H. Org. Lett.
2011, 13, 6378–6381.
(22) Buccella, D.; Horowitz, J.A.; Lippard, S.J. J. Am. Chem.
Soc. 2011, 133, 4101–4114.
(23) Iyoshi, S.; Taki, M.; Yamamoto, Y. Org. Lett. 2011, 13,
4558–4561.
(24) Du, P.; Lippard, S.J. Inorg. Chem. 2010, 49, 10753–10755.
(25) Tamanini, E.; Katewa, A.; Sedger, L.M.; Todd, M.H.;
Watkinson, M. Inorg. Chem. 2009, 48, 319–324.
(26) Shin, I.-S.; Bae, S.W.; Kim, H.; Hong, J.-I. Anal. Chem.
2010, 82, 8259–8265.
(17) Tomat, E.; Lippard, S.J. Curr. Opin. Chem. Biol. 2010, 14,
225–230.