ChemComm
Communication
4,40
5,50
6,60
7,70
gradually, while those for H , H , H , H were upfield shifted Then these cells were treated further with the cell membrane
Fig. S9, ESI†). The gradual broadening and shortening was also permeable metal chelator TPEN, and the fluorescence image
(
3
+
observed for all signals due to the paramagnetic nature of Fe . The showed that the initial cytosolic fluorescence was decreased
1
2,20
H NMR titration profile according to the alteration of H signal distinctly and only very few fluorescent spots were observed
3+
3+
suggests a 2 : 1 (Fe to L) Fe binding stoichiometry. Job’s plot based (Fig. 4c), suggesting that TPEN is able to remove the deposited
on the emission at 425 nm also confirmed the 2 : 1 Fe binding exogenous Fe effectively, while L is able to imaging Fe
stoichiometry (Fig. S10, ESI†). Moreover, the ESI-MS spectrum of L reversibly. The SKOV-3 cells without the overloaded Fe showed
solution mixed with 4 equiv. of Fe displays a main signal of m/z very weak fluorescence under the same imaging conditions
3+
3+
3+
3
+
3+
+
3+
8
06.67, which can be assigned as [L + 2Fe–4H] , besides the other (Fig. S14, ESI†). In addition, the higher Fe concentration for
signals for L (Fig. S11, ESI†). The apparent dissociation constant (K cell incubation leads to the higher fluorescence in cells (Fig. S15,
d
)
ꢁ
10
3+
was determined to be 5.61 ꢂ 10 M by fitting the fluorescent titration ESI†). The imaging ability of L in the Fe -overloaded HeLa cells
11
3+
profile (Fig. S12, ESI†). With the paramagnetic nature of Fe , the has also been investigated (Fig. S16 and S17, ESI†). All the results
3
+
turn-on Fe fluorescent response might be ascribed partially to either confirmed that L has excellent cell membrane permeability and
3
+
3+
3+
the low spin configuration of Fe or the electronic decoupling of Fe
a rapid/reversible cytosolic Fe imaging ability, which is its
3+
3+
from anthracene. Since Fe (15 equiv.) does not alter the emission of advantage over normal turn-on Fe chemodosimeters. With this
L , the analogue of L without the two hydroxyethyl pendants, in the reversible imaging ability, L can be applied to monitor the
0
12
3
+
2+
same Tris-HCl buffer (Fig. S13, ESI†), the hydroxyethyl pendants in L fluctuation of intracellular Fe . In addition, overloading Fe
0
should play an essential role in the turn-on sensing behavior of L. L into SKOV-3 cells did not lead to the enhanced fluorescence
3
+
14
displays a distinctly lower Fe binding ability than L and no mass inside the cells under the same imaging conditions (Fig. 4d–f),
0
3+
3+
3+
2+
signal for L –Fe complex was detected. Therefore, the effective Fe
binding ability of L might also contribute to its turn-on response.
implying that L is able to discriminate Fe from Fe inside the
cells. The fluorescence intensity in the regions of interest in
3
+
3+
With the reversible turn-on response to Fe and the Fig. 4 gives more distinct evidence of the turn-on Fe response
pH-independent fluorescence in pH 6.5–12.0, L might be a and Fe /Fe discriminating ability of L (Fig. S18, ESI†). The
potential imaging agent for Fe in live systems. The intracellular MTT assay shows that L has no distinct cytotoxicity against HeLa
Fe imaging ability of L was investigated in the Fe -overloaded and SKOV-3 cells (cell viability over 97% for HeLa cells, 93% for
SKOV-3 and HeLa cells using a confocal microscope (Zeiss LSM SKOV-3 cells) after 24 h of incubation at the level of 10 mM, which
10). Exogenous Fe was deposited into the live cells via ferric is higher than the cell staining concentration (Fig. S19, ESI†).
citrate incubation using a reported procedure (50 mM, 4 h,
7 1C). After removing the extracellular Fe via PBS washing was devised via integration of the Fe -chelating HEDTA with
3 times), the SKOV-3 cells were incubated with L (8.7 mM) for anthracene. Besides the appealing turn-on response specifically to
0 min. The confocal image displays a bright punctated fluores- Fe , its reversible and instant Fe response makes this sensor
3
+
2+
3
+
3
+
3+
3
+
7
3+
In summary, a [2+2] macrocycle-based Fe fluorescent sensor
1
3
3+
3+
3
(
3
3+
3+
3+
cence pattern in cytosol (Fig. 4b), while the nucleus is dim. more helpful than normal chemodosimeters for Fe . Moreover,
3
+
2+
the Fe /Fe transition at the mM level can be visualized using this
sensor upon irradiation. Preliminary study found that the hydro-
xyethyl pendants are essential for its turn-on response. Confocal
imaging disclosed that this sensor possesses not only the cell
membrane permeability but also the cytosolic Fe imaging
ability. The different fluorescent response to Fe and Fe of this
sensor has also been confirmed in live cells. This study provides
3
+
3+
2+
3
+
also the parent framework to construct the turn-on Fe sensor of
promoted imaging ability.
We thank the National Basic Research Program of China
(No. 2011CB935800) and National Natural Science Foundation
of China (No. 21271100, 21131003 and 91213305) for financial
support.
Notes and references
1 (a) P. Aisen, M. Wessling-Resnick and E. A. Leibold, Curr. Opin.
Chem. Biol., 1999, 3, 200; (b) M. D. Connie and C. W. Hsia, New Engl.
J. Med., 1998, 338, 239.
3
+
Fig. 4 Confocal fluorescence imaging of the Fe -overloaded (a–c) and
2+
Fe -overloaded (d–f) SKOV-3 cells stained by L. Bright-field transmission
3+
2+
image of cells incubated with 50 mM Fe (a) or Fe (d) for 4 h, followed by
PBS washing (3 times) and L incubation (8.7 mM in PBS buffer, 30 min,
2
(a) D. J. A. Netz, M. St u¨ mpfig, C. Dor ´e , U. M u¨ hlenhoff, A. J. Pierik
and R. Lill, Nat. Chem. Biol., 2010, 6, 758; (b) M. B. Murataliev,
R. Feyereisen and F. A. Walker, Biochim. Biophys. Acta, 2004, 1698, 1;
3
7 1C) in sequential; (b) fluorescence image of cells in (a); (c) fluorescence
image of cells in (b) followed by incubation with TPEN (50 mM, 30 min,
7 1C); (e) fluorescence image of cells in (d); (f) fluorescence image of cells
in (e) followed by the similar TPEN treatment. lex, 405 nm, band path 425–
60 nm. Scale bar, 10 mm. The fluorescence intensity of regions of interest
3
(
c) M. F. Lucas, D. L. Rousseau and V. Guallar, Biochim. Biophys.
Acta, 2011, 1807, 1305.
4
3 (a) R. S. Eisenstein, Annu. Rev. Nutr., 2000, 20, 627; (b) T. A. Rouault,
Nat. Chem. Biol., 2006, 2, 406.
was given in Fig. S18 (ESI†).
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 4631--4634 | 4633