A sulfonamidoquinoline-derived Zn2+ fluorescent sensor with 1:1 Zn2+ binding stoichiometry

Article abstract of DOI:10.1016/j.inoche.2010.11.021

The integration of bis(pyridin-2-ylmethyl)amine (BPA) with 8-sulfonamidoquinoline (SQ) resulted in a new fluorescent Zn²⁺ sensor of 1:1 binding stoichiometry. The synergic Zn²⁺ coordination of BPA and SQ motifs provides the sensor the advantage over TSQ and its analogues in discriminating mobile Zn²⁺ from the bound Zn²⁺ of unoccupied coordination sites in living systems. Its pH-independent Zn ²⁺-enhanced emission in physiological condition and cell permeability make it an effective intracellular Zn²⁺ imaging agent. This sensor profits also from its confirmed Golgi-preferential affinity, and its pH-independent Zn²⁺ response in physiological pH range provides it the advantages over other xanthenone-based sensors.

Full text of DOI:10.1016/j.inoche.2010.11.021

Inorganic Chemistry Communications 14 (2011) 304–307
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
A sulfonamidoquinoline-derived Zn2+ fluorescent sensor with 1:1 Zn2+
binding stoichiometry
Changli Zhang a,b, Yuming Zhang a,c, Yuncong Chen a, Zhijun Xie a, Zhipeng Liu a, Xindian Dong a,
a
a,
Weijiang He a, , Chen Shen , Zijian Guo
⁎
⁎
a
State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
Department of Chemistry, Nanjing Xiaozhuang College, Nanjing 210017, PR China
Xingling College, Nantong University, Nantong 226009, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The integration of bis(pyridin-2-ylmethyl)amine (BPA) with 8-sulfonamidoquinoline (SQ) resulted in a new
fluorescent Zn2+ sensor of 1:1 binding stoichiometry. The synergic Zn2+ coordination of BPA and SQ motifs
provides the sensor the advantage over TSQ and its analogues in discriminating mobile Zn2+ from the bound
Zn2+ of unoccupied coordination sites in living systems. Its pH-independent Zn2+-enhanced emission in
physiological condition and cell permeability make it an effective intracellular Zn2+ imaging agent. This
sensor profits also from its confirmed Golgi-preferential affinity, and its pH-independent Zn2+ response in
physiological pH range provides it the advantages over other xanthenone-based sensors.
© 2010 Elsevier B.V. All rights reserved.
Received 1 October 2010
Accepted 17 November 2010
Available online 25 November 2010
Keywords:
Sulfonamidoquinoline
Zinc
Fluorescent sensor
Imaging
Mobile Zn2+ is an important transmitter in neural signal
transmission, and is also proposed to regulate the pathophysiology
of many severe neurological diseases [1]. Mobile Zn2+ is also involved
in the signal transduction process [2]. The spatial and temporal Zn2+
tracking via fluorescent sensor staining is of great significance for
clarifying the mobile Zn2+-involved physiological process [1,3]. TSQ
(6-methoxy-(8-p-toluene sulfonamido)quinoline) and its analogue
Zinquin have been widely adopted to image mobile Zn2+ in cells and
tissues [4]. These classical Zn2+ fluorescence sensors exhibit the
distinct Zn2+-triggered “turn-on” fluorescence, cell permeability and
favorable pH-independence emission. Although many Zn2+ fluores-
cent sensors have been reported [5], the diversified microenviron-
ments in living systems demand sensors of different properties, and
modification of 8-sulfonamidoquinoline (SQ) is still an attractive
strategy [6]. However, the reported SQ-derived sensors are difficult in
discriminating mobile Zn2+ from the bound Zn2+ of unoccupied
coordination sites in living systems, since these bidentates require
extra ligand to stabilize their Zn2+ complexes [5,6] . In fact, their Zn2+
binding stoichiometry is 2:1 (Sensor/Zn2+). Modifying SQ-derived
sensors with additional synergic Zn2+ coordination sites to acquire
1:1 Zn2+ binding stoichiometry should be helpful to overcome this
disadvantage. In this study, a new Zn2+ fluorescent sensor, BPSQ, was
constructed for this purpose by combining SQ with the synergic zinc
coordinating motif, bis(pyridin-2-ylmethyl)amine (BPA) (Scheme 1).
The spectroscopic study of BPSQ was investigated in HEPES buffer
(pH, 7.2) containing 5% DMSO except for the pH titration. BPSQ (10 μM)
exhibits anintensiveabsorption bandcenteredat302 nm(Fig. 1a). Zn2+
titration demonstrates the linear decrease of this band and linear
increase of a new band centered at 360 nm, when the [Zn2+
]total/[BPSQ]
ratio being lower than 1:1. The stable spectra thereafter and the clear
isosbestic pointat326 nmsuggest thatonly oneZn2+ complex species is
formed. The titration profiles according to the absorbance at 302 and
360 nm suggest this complex possess the Zn2+ binding stoichiometry of
1:1.
BPSQ in HEPES buffer exhibits a typical broad emission band of TSQ
and analogues, centering at 532 nm with a shoulder peak at 497 nm
(λex, 364 nm). The pH titration demonstrates the stable emission from
pH 6.0 to 10.0, which makes BPSQ suitable for application in
physiological condition (Fig. S6). However, the higher pH above 10
leads to the distinct emission enhancement, suggesting the
⁎
Corresponding authors. Fax: +86 25 83314502.
E-mail addresses: heweij69@nju.edu.cn (W. He), zguo@nju.edu.cn (Z. Guo).
Scheme 1. Synthesis of BPSQ.
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.11.021

C. Zhang et al. / Inorganic Chemistry Communications 14 (2011) 304–307
305
BPSQ displays the distinct linear emission enhancement (Fig. 1b), and
(a)
the emission become stable when the [Zn2+
]total/[BPSQ] ratio being
0.5
0.4
0.3
0.2
0.1
0.0
higher than 1:1. The dissociation constant (Kd) of Zn2+/BPSQ complex
was determined to be (1.8 0.1)×10−12 M via fluorescence titration
in a series of Zn2+ buffer [7]. The limit of detection (LOD) for Zn2+ is
~4.2×10−14 M (3σ/slope, see supporting information). The quantum
3.0
2.5
2.0
1.5
1.0
360 nm
302 nm
[Zn2+]
yield of BPSQ and its Zn2+ complex is determined as 0.015 (ε364
,
420 M−1 cm−1) and 0.055 (ε364, 4680 M−1 cm−1) with quinine
sulfate solution (Φ=0.546, 0.5 M H2SO4) as reference. On the other
hand, single crystal structural analysis of its zinc complex exhibits a
1:1 Zn2+ binding stoichiometry of BPSQ. In this complex, Zn2+ center
adopts a distorted octahedral geometry with three BPA N atoms and
one quinoline N atom occupying the equatorial plane vertexes. The
two axial sites are occupied by the sulfonamide N atom and a water O
atom. The quinoline plane is almost perpendicular to the BPA pyridine
planes (Fig. 2). Structural analysis demonstrates the direct Zn2+
coordination of sulfonamide N via deprotonation. In addition, 1H NMR
Zn2+ titration of BPSQ displays the distinct changes in spectrum upon
Zn2+ addition, and the spectrum becomes stable when [Zn2+]/[BPSQ]
being higher than 1:1 (Fig. S9). The signal of H8 undergoes a distinct
downfield shift from 8.77 to 9.06 ppm upon Zn2+ addition, implying
that the quinoline N should be involved directly in Zn2+ coordination.
In addition, this coordination still induces the signal shifts of other
protons from the N-containing ring of quinoline. The Zn2+ coordina-
tion-induced sulfonamide deprotonation induces also the signal shift
of protons from another quinoline ring. Zn2+ coordination to BPA
motif in solution has also been confirmed by the 1H NMR monitoring
(Table S3, Fig. S9). 1H NMR titration suggests that the solution
coordination structure is almost identical to the crystal structure.
Therefore, the Zn2+-induced emission enhancement should have the
similar origin of other SQ-based Zn2+ sensors: Zn2+ coordination to
sulfonamide and quinoline N atom [6]. In addition, the PET (photo-
induced electron transfer) blockage via outer amine Zn2+ coordina-
tion may also induce the enhanced emission [3].
0.0
0.4
0.8
1.2
1.6
2+
[Zn ]/[BPSQ]
[Zn2+]
300
350
400
450
Wavelength (nm)
(b)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Cation-induced changes in emission enhancement factor F/F0 at
532 nm were investigated to determine the specific Zn2+ sensing
ability of BPSQ. Zn2+ and Cd2+ (1 equiv) induce F/F0 increase from 1
to 9.3 and 9.0, respectively. Considering the scarcity of Cd2+ in living
cells, the Cd2+ response should not interfere with intracellular Zn2+
detection. All other transition metal cations do not lead to any obvious
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
[S]/[S+Zn2+
]
(c)
8
7
6
5
4
3
2
1
0
6
5
4
3
2
1
0
emission enhancement. Although the pretreatment by Co2+, Cu2+
,
0.0 0.4 0.8 1.2 1.6 2.0
[Zn2+] /[BPSQ]
[Zn2+]
450
500
550
600
650
Wavelength (nm)
Fig. 1. (a) Absorption spectra of BPSQ (50 μM) in HEPES buffer (50 mM, 0.1 M KNO3, pH,
7.2, 5% DMSO) when titrated by Zn2+. Inset, the titration profiles based on A302 and A360
.
(b) Job's plot of BPSQ and Zn2+ in the same medium according to the absorbance at
360 nm. The total concentration of Zn2+ and BPSQ is 0.5 mM. S, BPSQ. (c) Emission spectra
of BPSQ (5 μM) in the same medium when titrated by Zn2+. Inset, the titration profiles
based on F532. λex, 364 nm. [Zn2+
]total increases along the direction of the arrow.
sulfonamide deprotonation favor the enhancement (pKa1 ~10.8).
Decreasing pH from 6.0 to 3.0 also results in the emission
enhancement due to PET blockage (pKa2 ~4.4). Zn2+ titration of
Fig. 2. Structural diagram of [Zn(BPSQ-H)(H2O)]NO3. The protons and anion are
omitted for clarification.

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C. Zhang et al. / Inorganic Chemistry Communications 14 (2011) 304–307
10
8
permeable chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylene di-
amine (TPEN) leads to the dim fluorescence (Fig. 4d). The mean
fluorescence intensity of three arbitrarily selected regions in nucleolus
(yellow panes) in Fig. 4b, c and d is 13, 118 and 25, respectively. For
the selected regions in cytoplasm (red panes), the mean intensity in
Fig. 4b, c and d is 8, 40 and 22. The preferential affinity of BPSQ to
certain organelles is also observed. Localization studies exhibit that
there is no colocalization when lysosome marker Red DND-99 is
costained by BPSQ (Fig. 4f). Partial colocalization (in yellow) is
observed when mitochondria marker Red CMXRos is costained by
BPSQ (Fig. 4g). There is still bright fluorescence (in green) of BPSQ that
is not overlaid. The further costaining experiment with Golgi marker
BODIPY TR Ceramide indicates that Golgi is the main preferential
organelle for BPSQ (Fig. 4h).
6
4
2
0
In conclusion, the newly developed Zn2+ fluorescent sensor BPSQ
displays not only the specific Zn2+-induced fluorescence response in
aqueous media, but also the 1:1 Zn2+ binding stoichiometry. This
stoichiometry provides this SQ-based sensor the ability to reduce the
interference in labile Zn2+ detection induced by the bound Zn2+ of
unoccupied coordination sites. The pH-independent fluorescence in
physiological condition, high Zn2+ selectivity/sensitivity and its cell
membrane permeability make this sensor an effective intracellular
Zn2+ imaging agent. Moreover, the current result displays a successful
modification of classic Zn2+ sensor as a new sensor of improved
imaging ability.
Fig. 3. Emission enhancement factor (F/F0 at 532 nm) of BPSQ (5 μM) in HEPES buffer
(50 mM, 0.1 M KNO3, pH, 7.2, 5% DMSO) induced by 1 equiv Zn2+, Cd2+, Co2+, Cu2+
,
Fe2+, Mn2+ and Ni2+ and 1000 equiv alkaline/alkaline earth metal cations. None-filled
bar, 1 equiv marked cation was added; sparsely-filled bar, the marked cation was added
after 1 equiv Zn2+; black bar, the marked cation was added before 1 equiv Zn2+
;
densely-filled bar, the marked cation and Zn2+ was added simultaneously. AM: all
metal cations including all the tested alkaline/alkaline earth metal cations (1000 equiv
each) and transition metal cations (1 equiv each) except Zn2+. S, BPSQ.
Fe2+ and Ni2+ (1 equiv) decreases the Zn2+-induced enhancement
factor from ~9.3 to ~4.0, the simultaneous addition of Zn2+ and all
other metal cations does not decrease this factor evidently (Fig. 3).
Acknowledgements
Moreover, the presence of 1000 equiv of Na+, K+, Ca2+ or Mg2+
,
which are abundant in cells, does not affect the Zn2+ sensing behavior
We thank the National Science Foundation of China (Nos.
20871066, 20631020, 90713001 20721002 and 10979019), National
Basic Research Program of China (No. 2011CB935800) and the Natural
Science Foundation of Jiangsu (BK2008015 and BK2009227) for the
financial support.
of BPSQ. On the other hand, the fluorescent Zn2+ sensing behavior of
BPSQ is not affected by different counter anions such as ClO−4 , Cl−
,
NO−3 , SO24−, and CH3CO−2 . All these suggest that BPSQ is a suitable
candidate for intracellular Zn2+-staining.
The intracellular Zn2+ imaging ability of BPSQ has been verified on
HeLa cells by confocal imaging. The imaging result discloses that the
stained HeLa cells exhibit dim inhomogeneous fluorescence (Fig. 4b).
When exogenous Zn2+ is introduced by incubation with 5 μM
pyrithione/ZnSO4, the cells display the bright punctated fluorescence
(Fig. 4c). The following deprival of the exogenous Zn2+ by cell
Appendix A. Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.inoche.2010.11.021.
Fig. 4. (a) Confocal microscopic images of HeLa cells stained by BPSQ (10 μM, 1× PBS, 20 min of incubation). λex, 405 nm. (b–d), fluorescence images via single channel imaging
(410–570 nm). (a), Bright-field transmission images; (b) images of BPSQ-stained cells; (c) fluorescence images of the rinsed cells in (b) followed by incubation with pyrithione/
ZnSO4 (1:1, 5 μM, 5 min) and BPSQ (10 μM, 20 min) in sequence; (d) fluorescence image of the cells in (c) followed by treatment with 50 μM TPEN (5 min). (e–h) Colocalization
images of cells incubated with 5 μM pyrithione/ZnSO4 (1:1) solution (5 μM, 5 min) followed by costaining with BPSQ and organelle dye. Image of cells stained by Hochest 33342
(5 μM, 15 min) and BPSQ (e), by LysoTracker Red DND-99 (50 nM, 15 min) and BPSQ (f), by MitoTracker Red CMXRos (50 nM, 15 min) and BPSQ (g), by Golgi marker BODIPY TR
Ceramide (5 μM, 30 min, 4 °C) and BPSQ (h).

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