Two-photon fluorescent probes of biological Zn(II) derived from 7-hydroxyquinoline

Article abstract of DOI:10.1021/ol901787w

A new fluorescent probe for monitoring Zn²⁺ was synthesized based on the structure of 7-hydroxyquinoline. Compared with 8-substituted quinolines, the new probe exhibited higher selectivity for Zn²⁺ over Cd²⁺. Its fluoresce

Full text of DOI:10.1021/ol901787w

ORGANIC
LETTERS
2
009
Vol. 11, No. 19
426-4429
Two-Photon Fluorescent Probes of
Biological Zn(II) Derived from
4
7-Hydroxyquinoline
†
,†
†
†
Xiao-Yun Chen, Jing Shi,* Yi-Ming Li, Feng-Liang Wang, Xu Wu,
‡
,†
,†,§
Qing-Xiang Guo,* and Lei Liu*
Department of Chemistry, UniVersity of Science and Technology of China,
Hefei 230026, China, Hefei National Laboratory for Physical Sciences at Microscale
and School of Life Science, UniVersity of Science and Technology of China,
Hefei 230026, China, and Department of Chemistry, Tsinghua UniVersity,
Beijing 100084, China
shijing@ustc.edu.cn; qxguo@mail.ustc.edu.cn; lliu@mail.tsinghua.edu.cn
Received August 3, 2009
ABSTRACT
A new fluorescent probe for monitoring Zn + was synthesized based on the structure of 7-hydroxyquinoline. Compared with 8-substituted
2
quinolines, the new probe exhibited higher selectivity for Zn + over Cd . Its fluorescence enhancement (14-fold) and nanomolar range sensitivity
2
2+
d
(K ) 0.117 nM) were favorable toward biological applications. Experiments also showed that a cell-permeable derivative of the new probe
was potentially useful for two-photon imaging in living cells.
Zn2 is involved in many biochemical processes, such as
+
because this method leads to less phototoxity, better three-
dimensional spatial localization, and greater penetration into
scattering or absorbing tissues. TPM is considered to be a
1
2
neurotransmission, cellular metabolism, enzyme regula-
3
4
2+
6
tion, and gene expression. Biological imaging of Zn ions
can provide direct information on their spatiotemporal dis-
good noninvasive means of fluorescence microscopy in tissue
5
7
tributions in living systems. In recent years, two-photon
explants and living animals. The application of the two-
microscopy (TPM) has gained much interest in biology
photon method to zinc physiology requires the parallel
development of novel zinc-specfic fluorescent probes that
†
Department of Chemistry, University of Science and Technology of
2
+
operate within living cells. Although various Zn probes
China.
‡
8
9
Hefei National Laboratory for Physical Sciences at Microscale and
have been designed based on fluorescein, coumarin, quino-
School of Life Science, University of Science and Technology of China.
10
§
line, and other fluorophores, only a few of them have been
used for two-photon imaging. The development of novel
Tsinghua University.
1
1
(
1) Burdette, A. C.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2003,
00, 3605.
2) Suhy, D. A.; Simon, K. D.; Linzer, D. I. H.; O’Halloran, T. V. J. Biol.
Chem. 1999, 274, 9183.
3) Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am.
1
(
(6) (a) Williams, R. M.; Zipfel, W. R.; Webb, W. W. Curr. Opin. Chem.
Biol. 2001, 5, 603. (b) Chang, C. J.; Nolan, E. M.; Jaworski, J.; Okamoto,
K.-I.; Hayashi, Y.; Sheng, M.; Lippard, S. J. Inorg. Chem. 2004, 43, 6774.
(c) Kim, H. M.; Kim, B. R.; Hong, J. H.; Park, J.-S.; Lee, K. J.; Cho, B. R.
Angew. Chem., Int. Ed. 2007, 46, 7445.
(
Chem. Soc. 2000, 122, 5644.
(
(
4) Falchuk, K. H. Mol. Cell. Biochem. 1998, 188, 41.
5) Que, E. L.; Domaille, D. W.; Chang, C. J. Chem. ReV. 2008, 108,
1
517.
(7) Terai, T.; Nagano, T. Curr. Opin. Chem. Biol. 2008, 12, 515.
10.1021/ol901787w CCC: $40.75
Published on Web 09/01/2009
 2009 American Chemical Society

two-photon excitied Zn2 fluorescent probes remains an
+
methoxy group at the 7-position of quinoline. We anticipate
that the change of the substitution position of the electron-
donating group may alter the ICT process and ion selectivity.
The synthesis of 7-MOQ is shown in Scheme 1 (details
are available in the Supporting Information). The X-ray
interesting and important challenge.
It is known that an efficient Zn2 probe for biological
applications should have sufficient water solubility, high
selectivity, high photostability, and long excitation wave-
+
1
2
length to avoid cell damage. Previously TSQ and Zinquin
have been found to be good Zn probes based on the
quinoline structure. To meet the demand of high selectivity
toward Zn , a strong chelator, i.e., di-2-picolylamine moie-
2
+
13
Scheme 1. Synthesis of 7-MOQ
2+
1
5
16
ty, was incorporated into quinoline. However, the ultra-
violet excitation wavelength (∼350 nm) of these quinoline-
14
based probes might damage living cells. This problem may
be solved with the TPM technique, which employs two
lower-energy, near-infrared photons for excitation.
We notice that most Zn2 probes based on the quinoline
structure poccess an oxygen or nitrogen atom at the 8-posi-
tion of quinoline, which participates in the coordiantion with
+
2
+
13,16
Zn .
It poses an interesting question as to whether the
2
+
sensing affinity and selectivity for Zn will be altered if
the substitution position changes. This change may also affect
the efficiency of molecular internal charge transfer (ICT)
of the chemosensor. Thus, we design and synthesize a novel
quinoline-derived Zn probe, 7-MOQ, which carries a
17
2
+
(
8) (a) Kikuchi, K.; Komatsu, K.; Nagano, T. Curr. Opin. Chem. Biol.
crystal structure of the zinc complex with 7-MOQ (Figure
1) demonstrates that the oxygen atom at the 7-position does
2
2
004, 8, 182. (b) Hirano, T.; Kikuchi, Y.; Nagano, T. J. Am. Chem. Soc.
002, 124, 6555. (c) Burdette, S. C.; Frederckson, C. J.; Bu, W.; Lippard,
S. J. J. Am. Chem. Soc. 2003, 125, 1278. (d) Nolan, E. M.; Jaworski, J.;
Okamoto, K.-I.; Hayashi, Y.; Sheng, M.; Lippard, S. J. J. Am. Chem. Soc.
2
005, 127, 16812. (e) Meng, X.-M.; Zhu, M.-Z.; Liu, L.; Guo, Q.-X.
Tetrahedron Lett. 2006, 47, 1559.
9) (a) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem.
(
Soc. 2007, 129, 13447. (b) Lim, N. C.; Yao, L.; Freake, H. C.; Bruckner,
C. Bioorg. Med. Chem. Lett. 2003, 13, 2251. (c) Lim, N. C.; Schuster, J. V.;
Porto, M. C.; Tanudra, M. A.; Yao, L.; Freake, H. C.; Br uˆ ckner, C. Inorg.
Chem. 2005, 44, 2018.
(
10) (a) Zhang, Y.; Guo, X.; Si, W.; Jia, L.; Qian, X. Org. Lett. 2008,
1
0, 473. (b) Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J. Am.
Chem. Soc. 2001, 123, 5160. (c) Hanaoka, K.; Kikuchi, K.; Kojima, H.;
Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 12470. (d) Zalewski,
P. D.; Forbes, I. J.; Borlinghaus, R.; Betts, W. H.; Lincoln, S. F.; Ward,
A. D. Chem. Biol. 1994, 1, 153. (e) Kimber, M. C.; Mahadevan, I. B.;
Lincoln, S. F.; Ward, A. D.; Tiekink, E. R. T. J. Org. Chem. 2000, 65,
8
204.
(
11) (a) Taki, M.; Wolford, J. L.; O’Halloran, T. V. J. Am. Chem. Soc.
2
004, 126, 712. (b) Bhaskar, A.; Ramakrishna, G.; Twieg, R. J.; Goodson,
T. III. J. Phys. Chem. C 2007, 111, 14607. (c) Kim, H. M.; Seo, M. S.; An,
M. J.; Hong, J. H.; Tian, Y. S.; Choi, J. H.; Kwon, O.; Lee, K. J.; Cho,
B. R. Angew. Chem., Int. Ed. 2008, 47, 5167.
Figure 1. X-ray crystal structure of the zinc complex of 7-MOQ.
(
12) (a) Joshi, B. P.; Cho, W.-M.; Kim, J.; Yoon, J.; Lee, K.-H. Bioorg.
Med. Chem. Lett. 2007, 17, 6425. (b) Jiang, W.; Fu, Q.; Fan, H.; Wang,
W. Chem. Commun. 2008, 259.
(
13) (a) Fahrni, C. J.; O’Halloran, T. V. J. Am. Chem. Soc. 1999, 121,
2+
not participate in coordination with Zn , which differs from
1
1448. (b) Frederickson, C. J.; Kasarskis, E. J.; Ringo, D.; Frederickson,
1
3,16
R. E. J. Neurosci. Methods 1987, 20, 91. (c) Zalewski, F. D.; Forbes, I. J.;
Betts, W. H. Biochem. J. 1993, 296, 403. (d) Liu, Y.; Zhang, N.; Chen, Y.;
Wang, L.-H. Org. Lett. 2007, 9, 315.
the previously reported 8-substituted quinolines.
The
nitrogen atoms of the di-2-picolylamine moiety and quinoline
(
14) Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am.
2
ring participate in zinc coordinations. One H O molecule is
Chem. Soc. 2000, 122, 5644.
15) (a) Wong, B. A.; Friedle, S.; Lippard, S. J. J. Am. Chem. Soc. 2009,
31, 7142. (b) Burdette, S. C.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.;
2+
also found to chelate to Zn to complete the five-coordina-
tion geometry.
(
1
1
Lippard, S. J. J. Am. Chem. Soc. 2001, 123, 7831. (c) Nolan, E. M.; Burdette,
S. C.; Harvey, J. H.; Hilderbrand, S. A.; Lippard, S. J. Inorg. Chem. 2004,
The H NMR spectra of 7-MOQ (Figure 2) indicate that
2+
6
upon coordination to Zn in DMSO-d the protons at the
4
3, 2624.
(
16) (a) Xue, L.; Wang, H.-H.; Wang, X.-J.; Jiang, H. Inorg. Chem.
ortho-position of pyridines shift downfield from 8.50 to 8.72
ppm. The proton at the 3-position of quinoline also shifts
downfield, suggesting the interaction between the fluorophore
2
008, 47, 4310. (b) Wang, H.-H.; Gan, Q.; Wang, X.-J.; Xue, L.; Jiang, H.
Org. Lett. 2007, 9, 4995. (c) Xue, L.; Liu, C.; Jiang, H. Org. Lett. 2009,
1
1, 1655.
2
+
(
17) (a) Sumalekshmy, S.; Henary, M. M.; Siegel, N.; Lawson, P. V.;
and Zn .
Wu, Y.; Schmidt, K.; Br e´ das, J.-L.; Perry, J. W.; Fahrni, C. J. J. Am. Chem.
Soc. 2007, 129, 11888. (b) Qian, F.; Zhang, C.; Zhang, Y.; H. W.; Gao,
X.; Hu, P.; Guo, Z. J. Am. Chem. Soc. 2009, 131, 1460.
The spectroscopic properties of 7-MOQ were measured
in aqueous buffer (25 mM HEPES, 0.1 M NaClO
4
, 5% v/v
Org. Lett., Vol. 11, No. 19, 2009
4427

ously reported 8- methoxylated Zn2 sensors (4-6-fold). The
+
2
+
d
apparent dissociation constant (K ) of 7-MOQ for Zn is
calculated to be 0.117 nM (Figure S5, Table S1, Supporting
Information). This value indicates that the probe can be used
in the subnanomolar range, which affords sufficient sensitiv-
1
9
ity for application in living cells.
To check the pH effect on the fluoresence response, the
2
+
fluorescence spectra of 7-MOQ with saturating Zn under
different pH conditions were examined (Figure S4, Support-
ing Information). It is found that the fluorescence intensity
remains constant for pH > 5. Therefore, the probe can be
used to monitor intracellular Zn without being affected by
the physiological pH change.
2+
Figure 2 1
.
H NMR spectra of the 7-MOQ (A) and 7-MOQ with
2
+
1
6
.0 equiv of Zn in DMSO-d (B).
The titration of 7-MOQ with different metal cations was
conducted to examine the selectivity of the probe (Figure
2
+
2+
+
DMSO, pH 7.4, 25 °C). The UV-vis absorption spectrum
of 7-MOQ exhibits a maximum absoption at 236 nm (Figure
S1, Supporting Information). As shown in Figure 3, free
4). Because Ca , Mg , and K are abundant in the bio-
Figure 4
. Fluorescence responses of 7-MOQ upon additions of
various metal ions. Experimental conditions: 5 µM 7-MOQ, 1.0
2
+
2+
+
2+
2+
2+
mM for Mg , Ca , and K , and 5 µM for Mn , Fe , Co ,
2
+
2+
2+
Figure 3
.
Fluorescence responses (λex ) 320 nm) of 5 µM 7-MOQ
Pb , Cu , and Cd , λ ) 320 nm.
ex
2+
upon the addition of Zn (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.6 equiv)
4
in the HEPES buffer (25 mM HEPES, 0.1 M NaClO , 5% (v/v)
DMSO, pH ) 7.4, I ) 0.1).
logical systems, they were tested at a concentration as high
as 1 mM. To our delight, these cations do not produce an
appreciable change in the fluorescence emission. Therefore,
the probe can be used under biological conditions even with
an increase of Ca concentration. The change of fluo-
rescence emission intensity was also not observed upon the
7
-MOQ showed weak fluorescence emisssion at 393 nm
4
-1
-1
upon excitation at 320 nm (ε ) 3.09 × 10 M cm , Φ
0
2
+
2+
)
0.039). Upon addition of Zn (0-1.6 equiv), the emission
intensity increases significantly. This enhancement can be
explained by the blocking of the photoinduced electron-
2
+
2+
addition of Mn and Pb . Other first row transition metal
2
+
2+
2+
18
ions including Fe , Co , and Cu might form complexes
with 7-MOQ and quench the fluorescence, but their influence
in vivo can be neglected due to their low concentration. Only
transfer (PET) pathway of 7-MOQ due to the binding with
2
+
Zn .
The maximum fluorescence emission intensity was ob-
2
+
2+
2
+
Zn and Cd induce the enhancement of fluorescence
tained when the Zn concentration increased to 1.0 equiv
Zn ) 0.556). A Job’s plot (Figure S3, Supporting Informa-
tion) also indicates the 1:1 binding model between 7-MOQ
2+
emission intensity. The interference of Cd is a well-known
problem of zinc fluorescence probes.
(Φ
8
,10,16
Compared to the
2
+
previous probes, 7-MOQ exhibits a much less pronounced
and Zn . It is important to point out that the fluorescence
2
+
interference by Cd .
enhancement (14-fold) of 7-MOQ is higher than the previ-
According to the above experiments, the methoxy group
at the 7-position does not participate in the coordination with
the zinc ion. To enhance the cell permeability the methoxy
(
18) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
7, 1515. (b) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima,
H.; Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc.
004, 126, 14079. (c) Wang, Y.-H.; Zhang, H.-M.; Liu, L.; Liang, Z.-X.;
Guo, Q.-X.; Tung, C.-H.; Inoue, Y.; Liu, Y.-C. J. Org. Chem. 2002, 67,
429. (d) Salman, H.; Tal, S.; Chuvilov, Y.; Solovey, O.; Abraham, Y.;
Kapon, M.; Suwinska, K.; Eichen, Y. Inorg. Chem. 2006, 45, 5315.
9
2
(19) (a) Nasir, M. S.; Fahrni, C. J.; Suhy, D. A.; Kolodsick, K. J.; Singer,
C. P.; O’Halloran, T. V. J. Biol. Inorg. Chem. 1999, 4, 775. (b) Hirano, T.;
Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2000,
122, 12399.
2
4428
Org. Lett., Vol. 11, No. 19, 2009

group was converted into dodecyloxy (7-DOQ). A prelimi-
nary study of 7-DOQ in living cells (A431) was carried out
by using confocal microscopy (Figure 5). Two-photon
level. These results indicate that 7-DOQ is cell permeable
2
+
and suitable for TPM imaging of intracellular Zn flux.
Cytotoxicity is a potential side effect of the probe that
must be controlled when dealing with living cells. To this
end, we have conducted MTT assays in HeLa cells. The cell
viability remains 90% after treatment with 7-DOQ (30 µM)
for 24 h. This cytotoxicity test indicates that low-micromolar
concentrations of 7-DOQ are essentially nontoxic for at least
2
4 h incubation and can be safely used for two-photon
bioimaging.
In conclusion, a novel 7-substituted, quinoline-based
fluorescent probe is designed and synthesized. It displays a
2
+
high selectivity and sensitvity for Zn in a neutral buffer
aqueous solution. Because the oxygen atom at the 7-position
does not participate in the coordination with zinc ion, the
new probe shows different properties as compared to the
2+
previously reported 8-substituted, quinoline-based Zn sen-
sors. The new probe exhibits a 14-fold fluorescence enhance-
2
+
ment in response to Zn . It has a dissociation constant of
2
+
2+
0
.117 nM with Zn and a higher selectivity toward Zn
2
+
over Cd . Furthermore, we demonstrate that a cell-perme-
able derivative of the new probe can be used for imaging
Zn in living cells with two-photon microscopy.
Figure 5
-DOQ after 30 min of incubation, λex ) 800 nm. (B) TP image
after a 30 min treatment with zinc(II)/pyrithione (50 µM, 1:1 ratio).
C) The overlay of (A) and (B). (D) TP image of cells that are
. (A) Bright-field image of A431 cells labled with 30 µM
7
2
+
(
further incubated with 50 µM TPEN for 10 min.
Acknowledgment. This work was supported by the
National Natural Science Foundation of China (Grant No.
9
0713009) and the Scientific Research Starting Foundation
for Returned Overseas Chinese Scholars, Ministry of Educa-
tion, China (Grant No. [2008]890).
microscopy (TPM) imaging of the 7-DOQ-labled cell
displays a very weak intracellular staining after incubation
at 37 °C for 30 min. The system then exhibits a strong blue
fluorescence with the addition of 1:1 zinc(II)/pyrithione (50
µM). Subsequent treatment with the metal ion chelator TPEN
Supporting Information Available: Experimental details
and compound characterizations. This material is available
free of charge via the Internet at http://pubs.acs.org.
(
50 µM) reverses the fluorescence intensity to the baseline
OL901787W
Org. Lett., Vol. 11, No. 19, 2009
4429