8-Aminoquinoline-based ratiometric zinc probe: Unexpected binding mode and its application in living cells

Article abstract of DOI:10.1016/j.bmcl.2013.04.042

PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn²⁺-induced red-shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoichiometry in PMQA-Zn²⁺, PMQA-Cu²⁺ shows 1:1 composition. Both nitrogen atoms from the aminoquinoline are missing in binding of zinc, while they are critically involved in Cu²⁺ chelation. The structure difference between PMQA-Zn²⁺ and PMQA-Cu²⁺ might shed light in designing novel zinc probes without suffering from copper interference.

Full text of DOI:10.1016/j.bmcl.2013.04.042

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3511–3514
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
8-Aminoquinoline-based ratiometric zinc probe: Unexpected binding
mode and its application in living cells
⇑
Liangwei Zhang, Xuemei Cui, Jinyu Sun, Yuli Wang, Weishuang Li, Jianguo Fang
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn2+-induced red-
shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoi-
chiometry in PMQA–Zn2+, PMQA–Cu2+ shows 1:1 composition. Both nitrogen atoms from the aminoquin-
oline are missing in binding of zinc, while they are critically involved in Cu2+ chelation. The structure
difference between PMQA–Zn2+ and PMQA–Cu2+ might shed light in designing novel zinc probes without
suffering from copper interference.
Article history:
Received 13 March 2013
Revised 15 April 2013
Accepted 17 April 2013
Available online 24 April 2013
Keywords:
Zinc probe
Ó 2013 Elsevier Ltd. All rights reserved.
8-Aminoquinoline
Living cell imaging
Zinc, the second most abundant d-block metal ion in human
body, is an essential element and plays multiple roles in various
biological processes such as maintaining brain function, regulating
gene expression, and keeping mammalian reproduction.1 Distur-
bance of the zinc homeostasis in living organisms is critically
related to a series of disease.2 Therefore, the detection of zinc has
attracted increasing interests.3 Among numerous methods, fluores-
cent sensors, due to the simplicity, high sensitivity, and super
biocompatibility, are a powerful tool in detection of zinc both
in vitro and in vivo.3,4 Particularly, ratiometric fluorescent probes
permit signal rationing to detect target molecules by measuring
the ratio of fluorescence intensities at two different wavelengths,
which could be independent of environmental effects.4c,d,5 And
thus, the past decades have witnessed a fruitful acquisition from
different groups.3,4 However, a majority of these sensors suffer
from diverse drawbacks, such as selectivity, sensitivity, water sol-
ubility as well as cell membrane permeability, which prevent them
from further application in living cells or animals.
8-Aminoquinoline (AQ) was commonly employed as fluoro-
phore for metal ion sensors based on internal charge transfer
(ICT), which is one of the most important signaling mechanism
in designing metal ion sensors.5b,6 In this sense, our previous
probe, 2-(bis(2-(ethylthio)ethyl)amino)-N-(quinolin-8-yl)acetam-
ide (BEAQA), bearing N/S/S heteroatoms as a receptor had de-
signed and synthesized, and successfully applied in imaging both
exogenous and endogenous zinc in living HeLa cells.6f However,
the stability of the probe has harassed us for its broad application
since the two sulfide groups in the molecule are prone to oxidation
during storage. To surmount this shortage, a new fluorescent probe
PMQA (Scheme 1) was prepared. The synthesis of sensor PMQA is
depicted in Scheme 1 according to the reported procedures with
some modifications.5b The probe PMQA, obtained in two steps with
62.4% overall yield starting from readily available AQ, was fully
characterized (Fig. S1–S6).
The absorbance and emission spectra of PMQA were examined
by UV–vis absorbance and fluorescence titrations. Two bands
(ꢀ240 nm & ꢀ302 nm) were both red-shifted with the addition
of Zn2+ (0–1.0 equiv) in the absorption spectra, accompanying
three isosbestic points at 245, 280 and 325 nm (Fig. 1A). The
absorption spectra is saturated by 0.5 equiv of Zn2+ (inset in
Fig. 1A), which is different from our previous zinc probe BEAQA,
whose spectra gets saturated by 1 equiv of Zn2+. Fluorescence spec-
tra of Zn2+ titration were shown in Figure 1B. PMQA has a very
weak fluorescence emission at about 415 nm with excitation at
325 nm. Upon addition of Zn2+, it displays a 85 nm red-shift of
emission to 500 nm and a remarkable enhancement of green fluo-
rescence intensity with a concomitant decrease of the weakly
intrinsic fluorescence, displaying a typically ratiometric fluoresce
character. The quick changes of the emission wavelength as well
as intensity upon zinc addition can be explained by a nearly planar
molecule conformation and a large p-electron conjugation system
of PMQA–Zn2+ complex (vide infra), which further enhance the ICT
process. The inset in Figure 1B shows the fluorescence intensity ra-
tio (F500/F415) as a function of equivalent of Zn2+. Again, the values
of F500/F415 reach a plateau by ꢀ0.5 equiv of Zn2+. Both absorbance
and emission spectral titrations suggest PMQA might form a 2:1
complex with zinc. It is worth noting that this fluorescence titra-
tion experiment was performed in a pure aqueous solution, which
is improved from other probes determined in organic solvent
⇑
Corresponding author. Tel.: +86 931 8912500; fax: +86 931 8915557.
E-mail address: fangjg@lzu.edu.cn (J. Fang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.042

3512
L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3511–3514
Scheme 1. Synthesis of PMQA.
Figure 2. Metal ion selectivity of PMQA. Light grey bars: fluorescence responses of
PMQA (10 M) and upon the addition of different metal ions in TB. Dark grey bars
(red bars): fluorescence responses of PMQA (10 M) with different metal ions
followed by addition of Zn2+ (10
M), kex = 325 nm.
l
mixed solutions,5b,6a,c,e,7 and thus has potential application in bio-
logical imaging. The solubility of the probe PMQA in Tris–HCl
(50 mM, pH 7.4) buffer (TB) was determined to be more than
1 mM (3 mg/10 ml) by directly suspending the solid powder in
the aqueous solution.
l
l
The selectivity of PMQA toward different metal ions was inves-
tigated as shown in Figure 2. There is no significant influence by
addition of 10 equiv of NaCl, KClO4, CaCl2, MgCl2, Al(ClO4)3 or
1 equiv of AgClO4, Ni(ClO4)2, Fe(ClO4)3, Mn(ClO4)2, HgCl2, CrCl3,
Co(ClO4)2, Cu(ClO4)2, Zn(ClO4)2. Addition of Cd(ClO4)2 caused a
slight enhancement of the intensity (ꢀthreefold), as zinc and cad-
mium are in the same group of the periodic table and have similar
properties. Compared to a 15-fold increase of emission induce by
zinc, PMQA easily distinguishes zinc from cadmium. Also,
cadmium is an ultratrace element, and has extremely low concen-
tration in human bodies. Taken together, the trivial interference
caused by cadmium in vitro should not prevent the application
of probe 1 for imaging zinc in vivo. Next, we determined if PMQA
can recognize zinc when it coexists with other metal ions. As
shown in Figure 2, Co2+ and Cu2+ almost completely quench the flu-
orescene signal, while other ions have little interference. This is
probably due to the displacement of zinc by Co2+ and Cu2+ since
nitrogen-containing lignands usually have higher affinity for them.
Many zinc probes exhibited similarly unfavorable responses due to
the competition from those ions.6c,8,11 Copper and cobalt belong to
essential elements. However, their concentrations are usually more
than 10 times lower than that of zinc in vivo,1c and their influence
could be neglected. The probe PMQA is also a sensitive sensor for
other zinc metal sources demonstrated in Figure S7.
Figure 3. Fluorescence titration spectra of PMQA–Zn2+ complex (5
lM) in TB upon
addition of increasing concentrations of Cu(ClO4)2 (0–1.5 equiv calculated based on
the amount of PMQA). The inset shows decay of fluorescence intensity at 500 nm
with increasing [Cu2+], kex = 325 nm.
addition of Cu2+ (0–1.5 equiv calculated based on the amount of
PMQA), and complete quenching achieved when more than
1.0 equiv of Cu2+ was added (Fig. 3), indicating a 1:1 complex
formed between PMQA and Cu2+, which is consistent with its crys-
tal structure (vide infra).
To further determine the binding stoichiometry of PMQA–Zn2+
complex, we adapted the Job’s plot based on the fluorescence titra-
tion. The maximal fluorescence intensity appears at about 0.39 mol
fraction (Fig. S8), supporting that the PMQA–Zn2+ complex has 2:1
stoichiometry. The most convincing evidence was provided by the
crystal structure of PMQA complexed with zinc (Fig. 4A), which
unambiguously demonstrates that two molecules of PMQA in a
head-to-tail orientation, acting as a hexadentate ligand via N atoms
Quenching of emission by Cu2+ is a depressing yet very common
drawback for diverse zinc sensors, which is usually caused by dis-
placement of zinc from the sensors by copper.6a,6c,8b–f,9,11 Subse-
quently, we examined the fluorescence response of PMQA–Zn2+
complex toward Cu2+ in detail. Remarkable fluorescence quenching
of PMQA–Zn2+ complex was observed dose-dependently upon
Figure 1. Absorption (A) and fluorescence (B) titration spectra of PMQA (10
lM) in TB with increasing concentrations of Zn(ClO4)2 (0–1.0 equiv), inset: the titration profile
based on the absorbance ratio at A352 nm/A302 nm and the fluorescence intensity ratio at F500 nm/F415 nm, kex = 325 nm.

L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3511–3514
3513
Figure 4. The crystal structures of PMQA–Zn2+ complex (A) and PMQA–Cu2+ complex (B) (30% probability level for the thermal ellipsoid). ClOꢂ4 and H atoms are omitted for
clarity.
and carbonyl oxygen atoms, form a complex with one molecule of
Zn2+. We also obtained the crystal structure of PMQA–Cu2+ complex
(Fig. 4B), which has 1:1 stoichiometry, surprisingly different from
that of PMQA–Zn2+ complex. It seems that neither nitrogen atoms
within the AQ structure is essential in binding of zinc, while they
are critically involved in coordination of copper. Since copper is a
common and potent competitor of zinc to bind to zinc sensors,8b–
The favorable in vitro results eventually prompt us to deter-
mine if PMQA can be applied in imaging zinc in living cells.
The Zn2+ sensing and imaging were carried out in HeLa cells.
As depicted in Figure 5A, HeLa cells show no fluoresce. After
incubation with PMQA (20 lM) for 1 h (Fig. 5B), weak green
fluorescence appeared, presumably caused by the probe captur-
ing intracellular zinc from its native ligands since PMQA has
high affinity for zinc. As Zn2+ accumulation by co-treatment
with ZnCl2 and sodium pyrithione, a zinc ionorphore,6d a dras-
tic increase of green fluorescence in cells was observed
(Fig. 5C). Fluorescence images revealed that PMQA is a cell
membrane-permeable probe and suitable for imaging Zn2+ in
living cells.
the structure difference between PMQA–Zn2+ and PMQA–Cu2+
f,9
sheds light in design novel probes that sense zinc without suffering
from copper interference.
Dissociation constant Kd and detection limit were obtained to
be 7.02 ꢁ 10ꢂ12 M2 and 8.85 ꢁ 10ꢂ8 M by fluorescence titration
(Fig. S9) according to the reported method.10,11 We also deter-
mined the reversibility of PMQA in binding zinc. The response of
PMQA to Zn2+ can be reversed by N,N,N0,N0-tetrakis(2-pyridyl-
methyl)ethylenediamine (TPEN) or EDTA (Fig. S10), two known
zinc chelators used in reversibility studies.6h The reversibility of
coordination of zinc by PMQA indicates the probe might has less
toxicity for imaging zinc in living systems.
In addition to metal ion selectivity, an ideal probe for biological
applications should have stable fluorescence signals within a broad
pH range. Therefore, we determined the fluorescence intensity of
PMQA in the absence and presence of Zn2+ under various pH con-
ditions. As shown in Figure S11, the free ligand is almost nonfluo-
rescent within the pH range of 2–14. After addition of Zn2+, the
fluorescence signal increased gradually when pH >4, and was fairly
stable from pH 5.5 to pH 12.0, indicating the potential application
of the probe in biological systems.
Zn2+ is an indispensable metal ion in cells, and plays vital roles in
various physiological/pathophysiological processes. Although tre-
mendous improvements have been achieved by different
groups,3,4,8f,12 specific and sensitive detection of endogenous Zn2+
still remains a challenge. The probe PMQA described here shows
excellent water solubility, high sensitivity and selectivity over a pa-
nel of potential interfering ions. The emission signal of PMQA–Zn2+
is fairly constant within a broad range of pH. Furthermore, the
reversibility of PMQA to bind with zinc indicates that the probe will
not permanently perturb the homeostasis of Zn2+ in living cells
when applied for in vivo imaging. Indeed, the probe PMQA appears
no cytotoxicity when HeLa cells were incubated with 20 lM of
PMQA overnight (data not shown). Compared to our previously re-
ported probe BEAQA,6f this one exhibits improved stability. When
the stock solution of PMQA (0.1 mM in TB) is kept at room
Figure 5. Fluorescence images of HeLa cells. (A) Normal HeLa cells; (B) HeLa cells incubated with PMQA; (C) HeLa cells incubated with ZnCl2 and pyrithione, and then with
PMQA.

3514
L. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3511–3514
temperature for a week, there is no significant loss of PMQA deter-
mined by HPLC analysis (data not shown).
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.04.042.
Copper, a common and potent competitor of zinc for diverse
zinc probes, usually quenches the emission signal triggered by
zinc, which lays an obstacle in designing practically applicable zinc
sensors.8b–f,9,11 Cu2+ complexes with nitrogen donor ligands are
typically found to be more stable than Zn2+ complexes by several
orders of magnitude. Interestingly, we found that PMQA displays
a completely different binding mode with Zn2+ and Cu2+. As evi-
denced by the crystal structures, neither nitrogen atom within
the AQ structure is involved in binding of zinc, which is extraordi-
narily unusual to most reported AQ-based zinc sesors.6a–g,9 PMQA
forms a head-to-tail dimer, and thus acts as a hexadentate lignand
to coordinate with zinc. However, this two nitrogen atoms, to-
gether with other two nitrogen atoms forming a quadridentate li-
gand, seems critically involved in binding with Cu2+. This structure
difference provides instructive information for designing selective
zinc probes without suffering from interference cansed by copper.
We are currently performing synthesis of novel probes based on
the different binding modes of PMQA with Zn2+ and Cu2+, and ex-
pect to report the discoveries in due time.
In conclusion, we have developed a reversible, ratiometric, and
readily synthesized fluorogenic probe PMQA with broad pH stabil-
ity, excellent water solubility, and cell membrane permeability.
The probe displays good selectivity and sensitivity for Zn2+ over
other metal ions in a pure aqueous solution. Upon binding with
zinc, the probe emits bright green fluorescence based on ICT mech-
anism, and decreases its intrinsic emission simultaneously. We
also successfully applied the probe PMQA in imaging zinc in living
HeLa cells. More importantly, the different binding modes of the
probe with zinc and copper provides unprecedented information
for designing novel zinc sensors without suffering from interfer-
ence cansed by copper.
References and notes
1. (a) Frederickson, C. J.; Koh, J. Y.; Bush, A. I. Nat. Rev. Neurosci. 2005, 6, 449; (b)
Vallee, B. L.; Falchuk, K. H. Physiol. Rev. 1993, 73, 79; (c) Roat-Malone, R. M.
Bioinorganic Chemistry: A Short Course; Wiley-Interscience, 2007.
2. (a) Lyubartseva, G.; Lovell, M. A. Biofactors 2012, 38, 98; (b) Foster, M.; Samman,
S. Antioxid. Redox Signal. 2010, 13, 1549.
3. (a) Xu, Z.; Yoon, J.; Spring, D. R. Chem. Soc. Rev. 1996, 2010, 39; (b) Pluth, M. D.;
Tomat, E.; Lippard, S. J. Annu. Rev. Biochem. 2011, 80, 333; (c) Tomat, E.; Lippard,
S. J. Curr. Opin. Chem. Biol. 2010, 14, 225; (d) Que, E. L.; Domaille, D. W.; Chang,
C. J. Chem. Rev. 2008, 108, 1517; (e) Kikuchi, K.; Komatsu, K.; Nagano, T. Curr.
Opin. Chem. Biol. 2004, 8, 182; (f) Kikuchi, K. Chem. Soc. Rev. 2010, 39, 2048.
4. (a) Kawabata, E.; Kikuchi, K.; Urano, Y.; Kojima, H.; Odani, A.; Nagano, T. J. Am.
Chem. Soc. 2005, 127, 818; (b) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.;
Nagano, T. J. Am. Chem. Soc. 2005, 127, 10197; (c) Xu, Z.; Baek, K. H.; Kim, H. N.;
Cui, J.; Qian, X.; Spring, D. R.; Shin, I.; Yoon, J. J. Am. Chem. Soc. 2010, 132, 601;
(d) Carol, P.; Sreejith, S.; Ajayaghosh, A. Chem. Asian J. 2007, 2, 338; (e) Nolan, E.
M.; Lippard, S. J. Acc. Chem. Res. 2009, 42, 193.
5. (a) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129,
13447; (b) Zhang, Y.; Guo, X.; Si, W.; Jia, L.; Qian, X. Org. Lett. 2008, 10, 473.
6. (a) Xue, L.; Liu, Q.; Jiang, H. Org. Lett. 2009, 11, 3454; (b) Fahrni, C. J.; O’Halloran,
T. V. J. Am. Chem. Soc. 1999, 121, 11448; (c) Zhou, X.; Yu, B.; Guo, Y.; Tang, X.;
Zhang, H.; Liu, W. Inorg. Chem. 2010, 49, 4002; (d) Nolan, E. M.; Jaworski, J.;
Okamoto, K.-I.; Hayashi, Y.; Sheng, M.; Lippard, S. J. J. Am. Chem. Soc. 2005, 127,
16812; (e) Zhou, X.; Li, P.; Shi, Z.; Tang, X.; Chen, C.; Liu, W. Inorg. Chem. 2012,
51, 9226; (f) Zhang, L.; Duan, D.; Cui, X.; Sun, J.; Fang, J. Tetrahedron 2013, 69,
15; (g) Wang, H. H.; Gan, Q.; Wang, X. J.; Xue, L.; Liu, S. H.; Jiang, H. Org. Lett.
2007, 9, 4995; (h) Hanaoka, K.; Muramatsu, Y.; Urano, Y.; Terai, T.; Nagano, T.
Chem. Eur. J. 2010, 16, 568.
7. (a) Du, J.; Fan, J.; Peng, X.; Li, H.; Sun, S. Sens. Actuators, B: Chem. 2010, 144, 337;
(b) Wang, J.; Lin, W.; Li, W. Chem. Eur. J. 2012, 18, 13629; (c) Zhang, Z.; Wang, F.
W.; Wang, S. Q.; Ge, F.; Zhao, B. X.; Miao, J. Y. Org. Biomol. Chem. 2012, 10, 8640;
(d) Bhalla, V.; Roopa; Kumar, M. Dalton Trans. 2013, 42, 975; (e) Zhou, Y.; Kim,
H. N.; Yoon, J. Bioorg. Med. Chem. Lett. 2010, 20, 125; (f) Li, Y.; Shi, L.; Qin, L.-X.;
Qu, L.-L.; Jing, C.; Lan, M.; James, T. D.; Long, Y.-T. Chem. Commun. 2011, 47,
4361.
8. (a) Zhang, J.; Cui, H.; Hojo, M.; Shuang, S.; Dong, C. Bioorg. Med. Chem. Lett. 2012,
22, 343; (b) Joshi, B. P.; Cho, W. M.; Kim, J.; Yoon, J.; Lee, K. H. Bioorg. Med. Chem.
Lett. 2007, 17, 6425; (c) Buccella, D.; Horowitz, J. A.; Lippard, S. J. J. Am. Chem.
Soc. 2011, 133, 4101; (d) Huang, S.; Clark, R. J.; Zhu, L. Org. Lett. 2007, 9, 4999;
(e) Nolan, E. M.; Jaworski, J.; Racine, M. E.; Sheng, M.; Lippard, S. J. Inorg. Chem.
2006, 45, 9748; (f) Zhou, Y.; Li, Z. X.; Zang, S. Q.; Zhu, Y. Y.; Zhang, H. Y.; Hou, H.
W.; Mak, T. C. Org. Lett. 2012, 14, 1214.
Acknowledgments
9. Li, Z.; Zhang, L.; Wang, L.; Guo, Y.; Cai, L.; Yu, M.; Wei, L. Chem. Commun. 2011,
47, 5798.
10. Baruah, M.; Qin, W.; Vallée, R. A.; Beljonne, D.; Rohand, T.; Dehaen, W.; Boens,
N. Org. Lett. 2005, 7, 4377.
11. Chen, M.; Lv, X.; Liu, Y.; Zhao, Y.; Liu, J.; Wang, P.; Guo, W. Org. Biomol. Chem.
2011, 9, 2345.
12. (a) Kimura, E.; Aoki, S. Biometals 2001, 14, 191; (b) Nolan, E. M.; Lippard, S. J.
Inorg. Chem. 2004, 43, 8310; (c) Nolan, E. M.; Ryu, J. W.; Jaworski, J.; Feazell, R.
P.; Sheng, M.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 15517; (d) Masanta, G.;
Lim, C. S.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc. 2011, 133,
5698.
This work was supported by the National Natural Science Found
of China (21002047, J1103307), the Ministry of Education of China
(20100211110027), Lanzhou University (the Fundamental Re-
search Funds for the Central Universities, lzujbky-2012-59), and
the 111 project.
Supplementary data
Supplementary data (synthesis, experimental details, spectro-
copical data, and crystallographic data) associated with this article