Ying Zhou et al.
2 with 3 in acetonitrile gave the target compound 1 in a high
yield of 72%. The structure of 1 was characterized, and the
detailed experimental procedures of the new compound are
described in the Supporting Information.
To clarify the interaction of 1 with metal ions, the UV/Vis
absorption and fluorescence spectra of 1 were studied in
CH3CN/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer (0.02m, pH 7.4, 9:1 v/v). Cr3+, Cd2+, Hg2+
,
Na+, Co2+, Zn2+, Fe3+, Ag+, Ni2+, Cu2+, Pb2+, FÀ, ClÀ, BrÀ,
2À
À
IÀ, SO42À, PO43À, HPO4 , and H2PO4 were used to mea-
sure the selectivity of probe 1. All spectra were recorded
after three minutes upon addition of 10 equivalents of each
of these ions. As shown in the Supporting Information, com-
pound 1 exhibited an absorption band major centered at
347 nm with two peaks at 330 and 315 nm. Upon addition of
different metal ions, no clear shift in the absorption maxi-
mum could be found. It was revealed that 1 had no clear se-
lectivity in absorption towards the tested ions. Compound
1 displayed two distinct and well-known fluorescent spectra
of pyrene moieties in which a peak at 381 nm could be at-
tributed to the monomeric emission, whereas another peak
at 483 nm comes from the excimer formation.[7] In the fluo-
rescence tests, compared to other ions examined, only Hg2+
generated a significant “turn-on” fluorescence response of
the excimer peak at 490 nm with a fluorescence enhance-
ment up to 22-fold. (Figure 1). The presence of Ag+ resulted
in a threefold fluorescence enhancement of the monomer
peak at 381 nm. These results suggest that 1 has a higher se-
lectivity toward Hg2+ than the other ions.
Figure 1. A) Fluorescent emission spectra of 1 (2.0ꢁ10À5 m) in DMSO/
HEPES buffer (0.02m, pH 7.4, 9:1 v/v) with 10 equiv of Cr3+, Cd2+, Hg2+
2À
, Na+, Co2+, Zn2+, Fe3+, Ag+, Ni2+, Cu2+, Pb2+, FÀ, ClÀ, BrÀ, IÀ, SO4
,
PO43À, HPO42À, and H2PO4À. B) Fluorescence intensity of 1 (2.0ꢁ10À5 m)
at 490 nm after the addition of 10 equiv of selected ions in DMSO/
HEPES buffer (0.02m, pH 7.4, 9:1 v/v): a) Cr3+, b) Cd2+, c) Hg2+, d) Na+
, e) Co2+, f) Zn2+, g) Fe3+, h) Ag+, i) Ni2+, j) Cu2+, k) Pb2+, l) FÀ, m) ClÀ,
Figure 2 shows the fluorescence enhancement of 1 upon
addition of various amounts of HgACHTNURTGNEUNG(ClO4)2 in DMSO/
n) BrÀ, o) IÀ, p) SO42À, q) PO43À, r) HPO42À, s) H2PO4
.
À
HEPES buffer (0.02m, pH 7.4, 9:1 v/v). Upon titration with
Hg2+, the fluorescence of the monomer peak at 381 nm in-
creased slowly, whereas the excimer peak at 490 nm in-
creased sharply. With the addition of Hg2+ (10 equiv) to a so-
lution of 1, the intensity of the peak at 490 nm increased dis-
tinctly with 22-fold enhancement. The ratio for the intensi-
ties (I490/I381) ranged from 0.8 to 5, which confirmed the
pens through the NH in thiourea groups, instead of through
the thione groups. The main reasons for this binding pattern
are the large steric hindrances from mercury ions as well as
the thione groups. The centroid–centroid distance (z) for
these two pyrene rings is 5.99 ꢃ, and q=58.638. These data
are thoroughly in line with the description of edge-to-face
stacking,[11] which strongly supports the p interactions of
stacked pyrene–pyrene dimers.
stacking of pyrene in the presence of Hg2+
.
The linear dependence of the intensity ratio within the
equivalent range of the Hg2+ ion showed that 1 forms a 1:1
complex with Hg2+, the association constant (Ka) of which
was determined to be about 1.34ꢁ104 mÀ1 from the titration
experiments.[8] Moreover, the Jobꢂs plot (see the Supporting
Information) confirmed 1:1 stoichiometry for the 1-HgII
complex, which strongly supported the above conclusions.
The fluorescence intensity of 1 is linearly proportional to
Hg2+ concentrations of 0–1 mm, and a detection limit[9] as
low as 7.4ꢁ10À7 m concentration of Hg2+ was established by
using 1 with a signal-to-noise ratio of 3 (see the Supporting
Information).
In addition to obtaining the insights of the binding pattern
of 1 with Hg2+, theoretical calculations were carried out. We
used density functional theory (B3LYP/6-31G* level)[10] that
could describe the binding pattern and p–p interactions. As
shown in Figure 3B, in the presence of Hg2+, two thiourea
units of probe 1 are thoroughly separated. The binding hap-
Further NMR spectroscopic analysis also provided evi-
dence of the binding mode of 1-Hg2+ (Figure 3D). With the
addition of varying concentrations of HgACTHUNTRGNE(UNG ClO4)2 to
1 (0.5 mm) in [D6]DMSO, the splitting of the Ha and Hb
peaks as well as a shift of Hc and Hd could be observed. In
the D2O exchange experiments, the peaks of Hc and Hd
were confirmed to be the hydrogen atoms in the NH groups
in the binding sites (see the Supporting Information). All re-
sults showed that a sandwich-stacking bind mode was
formed in the 1-Hg2+ complex.[12] With the binding of Hg2+
,
the separated pyrene moieties were pulled close and formed
stacked pyrene–pyrene dimers, which further increased the
excimer fluorescence at 490 nm.
To demonstrate the feasibility of 1 for its application to in
vivo imaging, fluorescence imaging tests were carried out in
the cells of C. elegans and zebrafish. Two ethanol-fixed cell
lines (BEAS-2B and CHO) were tested using 1-Hg2+ as the
Chem. Asian J. 2014, 9, 744 – 748
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