A Zn2+ fluorescent sensor derived from 2-(pyridin-2-yl) benzoimidazole with ratiometric sensing potential

Article abstract of DOI:10.1021/ol802537c

A fluorescent Zn²⁺ sensor based on the 2-(2'-pyridinyl) benzoimidazole (2-PBI) fluorophore has been devised by incorporation with a Zn²⁺ ionophore, bis(pyridin-2-ylmethyl)amine. The sensor (PBITA) demonstrates a Zn²⁺-speci

Full text of DOI:10.1021/ol802537c

ORGANIC
LETTERS
A Zn2+ Fluorescent Sensor Derived from
2-(Pyridin-2-yl)benzoimidazole with
Ratiometric Sensing Potential
2009
Vol. 11, No. 4
795-798
Zhipeng Liu,† Changli Zhang,†,§ Yunling Li,† Zhengyi Wu,† Fang Qian,†
Xiaoliang Yang,† Weijiang He,*,† Xiang Gao,‡ and Zijian Guo*,†
State Key Laboratory of Coordination Chemistry, Coordination Chemsitry Institute,
School of Chemistry and Chemical Engineering, Nanjing UniVersitry,
Nanjing 210093, P. R. China, Animal Model Research Center, Nanjing UniVersity,
Nanjing 210061, P. R. China, and Department of Chemistry, Nanjing Xiaozhuang
College, Nanjing 210017, P. R. China
zguo@nju.edu.cn; heweij69@nju.edu.cn
Received November 19, 2008
ABSTRACT
A fluorescent Zn2+ sensor based on the 2-(2′-pyridinyl)benzoimidazole (2-PBI) fluorophore has been devised by incorporation with a Zn2+
ionophore, bis(pyridin-2-ylmethyl)amine. The sensor (PBITA) demonstrates a Zn2+-specific emission shift and enhancement with a 1:1 binding
ratio. Due to the Zn2+-induced coplanation of 2-PBI via reversion/rotation, PBITA is shown to behave as a ratiometric sensor. The intracellular
Zn2+ imaging ability of the sensor has been tested in HeLa cells using a confocal microscope.
As the second most abundant transition-metal ion in the
human body, Zn2+ is actively involved in various biological
processes.1 Spatiotemporal determination of Zn2+ in biologi-
cal samples utilizing fluorescent sensors is of great signifi-
cance for understanding the role of Zn2+ in biology. As a
consequence, development of novel fluorescent sensors for
Zn2+ has received considerable current attention.2-8 Most
of the currently reported Zn2+ fluorescent sensors have the
nature of metal chelation enhanced fluorescence (MCHEF),9
which functions via Zn2+ binding-induced emission enhance-
ment. Normally, the quantum yield of fluoresent sensors
displays distinct environment-dependence. This property
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† Coordination Chemsitry Institute.
‡ Animal Model Research Center.
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§ Nanjing Xiaozhuang College.
(1) Berg, J. M.; Shi, Y. Science 1996, 271, 1081–1085.
(2) Kimura, E.; Kioke, T. Chem. Soc. ReV. 1998, 27, 179–184.
10.1021/ol802537c CCC: $40.75
Published on Web 01/21/2009
2009 American Chemical Society

allows these MCHEF sensors to visualize the change of Zn2+
concentration, but they cannot provide quantified information
about [Zn2+]free. Ratiometric Zn2+ sensors are capable of
overcoming this problem because Zn2+ binding to them
induces shift in excitation or emission maxima, and internal
calibration can be achieved by measuring the ratio of apo-
sensor and Zn2+-bound sensor. Then, not only the environ-
ment-dependence but also the artifacts caused by the
variations in excitation intensity, emission collection ef-
ficiency, and photobleaching can be largely reduced by
internal calibration. However, ratiometric fluorescent Zn2+
sensors suitable for practical intracellular Zn2+ imaging are
rare so far,10 due to the scarcity of suitable fluorophore
prototypes displaying zinc chelation-induced emission/excita-
tion shift.10,11 Therefore, there is a huge scope and potential
for exploring novel fluorophores for ratiometric Zn2+ sensing.
A common fluorophore, 2-(2′-pyridinyl)benzoimidazole (2-
PBI), is widely used in coordination chemistry for its ability
to bind an array of d- and f-block elements. It is able to act
as both fluorophore and ionophore for Zn2+ and displays a
specific emission shift in both aqueous and acetonitrile
solution owing to Zn2+-chelation via 2, 2′-N atoms. However,
2-PBI fails to qualify as a ratiometric Zn2+ sensor due to
the low Zn2+ binding affinity and variable Zn2+ binding
modes.12 Increasing the Zn2+ coordination number of 2-PBI
could enhance the Zn2+ binding ability and define the Zn2+
binding mode, which will be favorable for the construction
of practical Zn2+ ratiometric sensors.
incorporated with 2-PBI at its 3′-position as the synergic Zn2+
coordination motif of its 2,2′-N atoms. Then, both 1:1 Zn2+
binding mode and higher Zn2+ binding affinity were ex-
pected. The synthetic procedure of PBITA is depicted in
Scheme 1. A refluxing ethanol solution containing o-
Scheme 1. Synthesis of PBITA
phenylenediamine and 6-(hydroxymethyl)pyridine-2-carbal-
dehyde in the presence of NaHSO3 afforded compound 1,
and PBITA was obtained with satisfactory yield by reacting
the tosylated derivative of 1 with BPA in CH3CN in the
presence of K2CO3 (Supporting Information).
The metal-binding behavior of PBITA has been deter-
mined by UV-vis and fluorescence spectroscopic studies.
Although PBITA is not highly water soluble, it can be
dissolved in water when 10% (v/v) of DMSO was added
and all the following studies were carried out in aqueous
solution containing 10% DMSO. This protocol is commonly
used in many reported Zn2+ sensors for intracellular Zn2+
imaging.9h-j,10b The UV-vis spectrum of PBITA in HEPES
buffer exhibits a maximal absorption band centered at 312
nm (ε ) 9.2 × 104 M-1 cm-1) with a shoulder band at 327
nm (Figure S4, Supporting Information). When titrated by
Zn2+ (0 - 2 equiv), the intensity of the maximal absorption
band decreased with the concomitant increase of the shoulder
band. The presence of a clear isobestic point implies the
conversion of free PBITA sensor to the only Zn2+ complex.
The titration profile can be drawn from the absorbance
changes at 312 nm, which suggests a 1:1 Zn2+ binding mode
of PBITA. The stoichiometry of the Zn2+/PBITA complex
has also been confirmed by mass spectroscopic determina-
tion. The electrospray ionization mass spectrum of this
complex displays two signals of m/z 235.16 and 469.25,
which can be assigned as the signals for [M + Zn - H]+
Herein, a fluorescent sensor derived from the 2-PBI
platform, PBITA, was prepared. In this compound, the Zn2+
chelator bis(pyridin-2-ylmethyl)amine (BPA) moiety was
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1
and [M + Zn]2+, respectively. The H NMR data provided
further evidence for the 1:1 binding ratio (Figure S7,
Supporting Information). All the aromatic and alkyl protons
of PBITA showed evident chemical shift changes in the
titration experiment, suggesting the involvement of 2,2′-N
atoms of 2-PBI and all the N atoms of the BPA motif in
Zn2+ coordination.
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Free PBITA in neutral HEPES buffer (DMSO/water )
1:9, v/v) exhibits weak fluorescence with two emission bands
796
Org. Lett., Vol. 11, No. 4, 2009

centered at 360 and 385 nm, respectively, with λex of 336
nm (Figure 1). The conformational change of PBITA (vide
Figure 1
.
Emission spectra of PBITA (1 × 10-5 M) obtained in
Figure 2. Emission ratio at 423 and 360 nm of PBITA (5 µM)
HEPES buffer (50 mM, DMSO/water ) 1:9, v/v, pH ) 7.2) when
titrated with Zn2+ (1 × 10-2 M). λex, 336 nm. The [Zn]total values
are 0, 1.5, 2.5, 4.0, 5.5, 6.5, 8.0, 9.0, 10.0, 11.0 µM (from bottom
to top). The inset is the corresponding Zn2+ titration profile
according the emission at 423 nm.
induced by different metal cations in HEPES buffer (50 mM, pH
7.2, 0.1 M KNO3, DMSO/water ) 1:9, v/v). λex, 336 nm. The final
concentration for Zn2+, Cu2+, Fe2+, Hg2+, Mn2+, Ni2+, and Pb2+ is
10 µM, for Na+, K+, Ca2+, and Mg2+ is 2 mM.
After incubation with PBITA solution (10 µM in PBS,
DMSO/water ) 1:9, v/v) at 25 °C for 20 min, the HeLa
cells displayed very faint intracellular fluorescence (Figure
3). However, HeLa cells exhibited intensive fluorescence
infra) should be responsible for the dual emission behavior
(Figure 1, bottom spectrum).13 Its quantum yield in neutral
buffer is 0.048 (Supporting Information). The titration of
Zn2+ into PBITA gave a new emission band centered at 423
nm which showed a linear enhancement with the increase
of [Zn2+]total when the ratio of [Zn2+]total/[PBITA] is below
or equal to 1:1. When the ratio reached 1:1, however, higher
[Zn2+]total did not lead to any further emission enhancement.
The remarkable bathochromic shift made PBITA a potential
ratiometric sensor for Zn2+. The emission band at 360 nm
also displayed some minor changes in intensity. When 1.0
molar equiv of Zn2+ was added, the ratio of the emission
intensity at 423 and 360 nm (F423/F360) increased from 0.77
(free PBITA) to 7.66 (Zn2+/PBITA complex). On the other
hand, the pH titration results of PBITA demonstrated that
F423/F360 varies slightly from 1.3 at pH 6.4 to 1.4 at pH 7.3,
which makes it suitable for application in physiological
conditions (Figure S8, Supporting Information).
The Zn2+-specific ratiometric response of PBITA was
further confirmed by screening the biologically relevant metal
cations. As shown in Figure 2, all tested metal cations except
Zn2+ did not induce any distinct emission shift and enhance-
ment. Moreover, the presence of Na+, K+, Ca2+, and Mg2+,
which are abundant in cells, did not interfere with the
ratiometric response to Zn2+, even though their concentration
was 1000 times higher than [Zn2+]total. A competitive binding
experiment gave an estimated Kd of 7.9 × 10-12 M for Zn2+/
PBITA complex (Figure S6, Supporting Information). The
quantum yield for the Zn2+/PBITA complex is 0.075.
Therefore, PBITA has the favorable property required for
intracellular Zn2+ imaging.
Figure 3. Confocal fluorescence imaging of HeLa cells: (a) bright-
field transmission image of cells labeled with PBITA (10 µM, PBS
solution containing 10% DMSO) at 25 °C for 20 min; (b)
fluorescence image of (a); (c) fluorescence image after incubation
with 5 µM ZnSO4/pyrithione (1:1) solution followed by rinse with
10 µM PBITA solution; (d) fluorescence image of HeLa cells in
(c) followed by further incubation with 50 µM TPEN solution for
20 min. λex, 356 nm. The transformation from light green to brown
denotes the emission enhancement. Bar ) 20 µm.
when exogenous Zn2+ was introduced into the cells via
incubation with ZnSO4/pyrithione solution. Moreover, the
intensive fluorescence was deeply depressed by scavenging
Zn2+ from the cells with the cell permeable metal chelator,
N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN).
These results indicate that PBITA is an effective intracellular
Zn2+ imaging agent with cell permeability. It also implies
that 2-(pyridin-2-yl)benzoimidazole could be an effective
model fluorophore to construct ratiometric sensors for Zn2+.
The intracelluar Zn2+ imaging behavior of PBITA on HeLa
cells was studied with a laser scanning confocal microscope.
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Org. Lett., Vol. 11, No. 4, 2009
797

The ratiometric sensing behavior of PBITA was further
investigated by molecular modeling. Two stable conforma-
tions of free PBITA optimized by density functional theory
(DFT) calculations at the B3LYP/6-31G(d,p) level (Gaussian
03)14 are shown in Figure 4. The proton attached to the
aryl planes via the reversion or rotation of a benzoimidazole
motif from the cis- (A) or trans-form (B). The polarized
moment of the PBITA molecule is distinctly changed in the
Zn2+ binding process, which results in the shift of absorption/
emission band. The blockage of the photoinduced electron
transfer (PET) process from a BPA amine to a 2-PBI
fluorophore induced by Zn2+ coordination to a BPA amine
should provide for Zn2+-induced emission enhancement.
In conclusion, a novel Zn2+ fluorescent sensor, PBITA,
demonstrates a Zn2+-specific ratiometric sensing behavior.
The incorporation of a BPA motif provides additional
synergic Zn2+ coordination sites, which gives exclusively
zinc complex with a 1:1 stoichiometry. The intracellular Zn2+
imaging ability on HeLa cells demonstrates that PBITA is
an effective Zn2+ imaging agent. Molecular modeling study
suggests that the Zn2+-induced red emission shift of PBITA
could be correlated to the coplanation of two heteroaromatic
planes of 2-PBI via Zn2+-induced reversion or rotation.
Current results indicate that the 2-PBI fluorophore and
analogues can be potentially applied for ratiometric Zn2+
sensing. Compared with the ratiometric sensors functioning
via Zn2+ binding induced-deprotonation, such as AQZ,11h
the ratiometric sensing behavior of the current sensor shows
lower relevance to the pKa value of sensor. The results
indicated that 2-PBI provides a valuable framework for the
construction of effective Zn2+-specific ratiometric sensors.
Figure 4. Conformations of PBITA optimized by density functional
theory calculations: free PBITA (A, B) and Zn2+/PBITA complex
(C). All of the protons except for the one attached to imidazole N
atom are omitted for clarity.
imidazole N atom locates respectively at the same side of
pyridine Na in conformation A (cis-form) and at the opposite
of pyridine Na in conformation B (trans-form). The dihedral
angle between the 2-pyridine plane and benzoimidazole plane
is 0° in A and 23.34° in B, respectively. The two stable forms
should be responsible for the dual emission of the sensor.
On the other hand, the structure of the Zn2+/PBITA complex
(C) was also optimized with an initial structure constructed
with direct Zn2+ coordination by three N atoms of BPA, two
2,2′-N atoms of 2-PBI, and one Cl-. The optimized structure
of the Zn2+/PBITA complex displays a dihedral angle of
0.93° between 1,1′-bridged aryl planes. Moreover, the proton
attached to the imidazole N atom points to the opposite
direction of pyridine Na. The theoretical study demonstrates
that Zn2+-binding leads to the coplanation of the 1,1′-bridged
Acknowledgment. We thank the National Science Foun-
dation of China (Nos. 20571043, 20631020, 90713001,
20871066, and 20721002) and the Natural Science Founda-
tion of Jiangsu Provence (BK2008015) for financial support.
We are grateful to the High Performance Computing Center
of Nanjing University for the award of CPU hours to
accomplish this work.
Supporting Information Available: Experimental details
and characterization of PBITA and selected fluorescence
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(14) Firsch, M. J. et al. Gaussian 03, Revision D.01; Gaussian, Inc.:
Wallingford, CT, 2004. For the full reference, see the Supporting Informa-
tion.
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Org. Lett., Vol. 11, No. 4, 2009