Design of ratiometric fluorescent probes based on arene-metal-ion interactions and their application to CdII and hydrogen sulfide imaging in living cells

Article abstract of DOI:10.1002/chem.201304181

Non-coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene-metal-ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg ^II, and Pb^II. X-ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene-metal-ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses. Copyright

Full text of DOI:10.1002/chem.201304181

DOI: 10.1002/chem.201304181
Full Paper
&
Fluorescent Probes
Design of Ratiometric Fluorescent Probes Based on Arene–Metal-
Ion Interactions and Their Application to CdII and Hydrogen
Sulfide Imaging in Living Cells
Ippei Takashima,[a] Miyuki Kinoshita,[a] Ryosuke Kawagoe,[a] Saika Nakagawa,[b]
Manabu Sugimoto,[b] Itaru Hamachi,[c] and Akio Ojida*[a]
Chem. Eur. J. 2014, 20, 2184 – 2192
2184
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Abstract: Non-coordinative interactions between a metal
ion and the aromatic ring of a fluorophore can act as a versa-
tile sensing mechanism for the detection of metal ions with
a large emission change of fluorophores. We report the
design of fluorescent probes based on arene–metal-ion in-
teractions and their biological applications. This study found
that various probes having different fluorophores and metal
binding units displayed significant emission redshift upon
complexation with metal ions, such as AgI, CdII, HgII, and PbII.
X-ray crystallography of the complexes confirmed that the
metal ions were held in close proximity to the fluorophore
to form an arene–metal-ion interaction. Electronic structure
calculations based on TDDFT offered a theoretical basis for
the sensing mechanism, thus showing that metal ions elec-
trostatically modulate the energy levels of the molecular or-
bitals of the fluorophore. A fluorescent probe was success-
fully applied to the ratiometric detection of the uptake of
CdII ions and hydrogen sulfide (H2S) in living cells. These re-
sults highlight the utility of interactions between arene
groups and metal ions in biological analyses.
Introduction
emission redshift has yet to be fully elucidated. In this article,
we report the design of ratiometric fluorescent probes based
on non-coordinative arene–metal-ion interaction. In this work,
we found that various types of fluorescent probes having the
different fluorophores, including anthracene, xanthene, and py-
ronine, exhibited significant redshifts in their emission upon
complexation with metal ions, such as AgI, CdII, HgII, and PbII.
In addition, we found that the emission redshift was also ob-
served with probes incorporating different metal-ion binding
units, allowing us to selectively detect specific metal ions. X-
ray crystallography and theoretical computational calculations
of AgI and CdII complexes revealed that non-coordinative
arene–metal-ion interactions (arene–metal-ion contact) be-
tween the fluorophore and the metal ions electrostatically
modulate the energy levels of the molecular orbitals of the flu-
orophore to cause the emission redshift. By exploiting large
emission shift within the visible wavelength region, our fluo-
rescent probe was successfully applied to the ratiometric visu-
alization of the toxic CdII ion uptake in living cell. In addition,
the CdII complex was applicable to in cell ratiometric imaging
of bioactive hydrogen sulfide (H2S). These are the first exam-
ples of the imaging applications using fluorescent probes
based on arene–metal-ion contact, highlighting the versatility
of this sensing mechanism in biological fluorescence analysis.
Fluorescent molecular probes that can selectively transform
chemical information into a detectable photosignal find use as
essential tools in many research areas. Of particular importance
is the use of fluorescent probes for in cell and in vivo bioimag-
ing, both of which have expanded dramatically in the last few
decades.[1] Over this period of time, particular interest has
been devoted to the fluorescent sensing of metal ions, an ex-
ample being the development of fluorescent probes for bio-
logically relevant metal ions, such as sodium(I), potassium(I),
and calcium(II).[2] Recently, various fluorescent molecular
probes capable of sensing zinc(II), copper(I), cadmium(II), and
mercury(II) ions have also been developed to allow the roles of
these transition and heavy metals in cell biology to be
studied.[3,4]
It is known that metal ions such as CdII and AgI induce a red-
shift in the emission of fluorophores by forming non-coordina-
tive arene–metal-ion interactions.[5] Czarnik and co-workers re-
ported in 1990[5b] the first definitive example of this phenom-
enon, in which an anthracene derivative with an azamacrocylic
ligand displayed a broad redshifted emission upon complexa-
tion with CdII in aqueous solution (0.1m CAPS buffer, pH 10).
Although this unique sensing mechanism could potentially
allow the design of new ratiometric fluorescent probes, the ap-
plication of arene–metal-ion interaction to fluorescence sens-
ing of metal ions and other substances is still very limited.[5] In
addition, the detailed mechanism of the metal-ion-induced
Results and Discussion
[a] I. Takashima, M. Kinoshita, R. Kawagoe, Prof. Dr. A. Ojida
Department of Pharmaceutical Sciences
Graduate school of Kyushu University
Design of fluorescent probes
The fluorescent probes used in this study are shown in
Figure 1. We employed three representative fluorophores (an-
thracene, xanthene, and pyronine) as the fluorescent scaffolds,
which have different emission wavelengths in the visible
region. These fluorophores were conjugated with two sets of
metal-ion binding sites; 2,2’-dipicolylamine (Dpa), iminodiace-
tate (Ida) or bis[2-(ethylthio)ethyl] amine (Beta). Anthracene
(1d) and xanthene (2d) derivatives, both of which had a single
Dpa site, were also prepared as control probes. The synthetic
procedures of these compounds are described in Schemes S1–
S3 in the Supporting Information and their fluorescence and
absorption properties are summarized in Table S1.
3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8152 (Japan)
Fax: (+81)926426601
E-mail: ojida@phar.kyushu-u.ac.jp
[b] S. Nakagawa, Prof. Dr. M. Sugimoto
Department of Science and Technology
Graduate school of Kumamoto University
2-39-1, Kurokami, Chuo-ku, Kumamoto 860-8555 (Japan)
[c] Prof. Dr. I. Hamachi
Department of Synthetic Chemistry and Biological Chemistry
Graduate School of Engineering, Kyoto University
Katsura, Nishikyo-ku, Kyoto 615-8510 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304181.
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2185
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
that the binding stoichiometry between 1a and the CdII ion is
1:1 (Figure S1 in the Supporting Information). In contrast, the
addition of CdII did not induce an emission shift in 1d, which
has a single Dpa site (Figure S2 in the Supporting Information).
This result implies that the two Dpa sites are essential to the
induction of an emission redshift on the formation of the
metal-ion complex. Fluorescence titrations of 1a were per-
formed with other metal ions to obtain further information of
the coordination-induced emission shift (Figure 2c). Among
the 11 metal ions investigated, CdII and AgI induced a signifi-
cant fluorescence emission redshift (Table 1 and Figure S6 in
the Supporting Information) while other metal ions scarcely in-
duced the emission redshift. To obtain structural information
of the metal complexes, X-ray crystallographic analyses of the
CdII, AgI, ZnII, and PbII complexes of 1a were carried out. The
resulting structure of the 1a–CdII complex is shown in Fig-
ure 2d and 2e. In this complex, the six nitrogen atoms of the
two Dpa sites are coordinated to the CdII ion, which is located
adjacent to the upper p-plane of the anthracene ring. The dis-
tance between the CdII ion and the nearest neighboring C9
atom is 3.14 ꢁ, which is smaller than the sum of the Van der
Waals radii of a CdII ion (1.58 ꢁ) and an aromatic carbon
(1.77 ꢁ). Figure 2e presents a cross-sectional view of the 1a–
CdII complex, which clearly shows the Van der Waals contact
(or arene–metal-ion contact) between the CdII ion and the C9
carbon. In the 1a–AgI structure (Figure 2 f and 2g), five of the
Dpa nitrogen atoms are coordinated to the AgI ion and the dis-
tance between the AgI ion and the C9 carbon atom is 3.11 ꢁ,
which again is less than the sum of the Van de Waals radii of
a AgI ion (1.72 ꢁ) and an aromatic carbon (1.77 ꢁ). In contrast,
metal-ion contact was not observed in the structures of the bi-
nuclear ZnII and PbII complexes of 1a (Figure S3 in the Sup-
porting Information), neither of
Figure 1. Structures of the fluorescent probes.
Metal-ion-induced emission shift of the anthracene probe
We first examined the change in the emission of the anthra-
cene derivative 1a upon complexation with CdII, based on
a prior report by Czarnik and co-workers.[5b] As shown in Fig-
ure 2a, the emission of 1a was shifted from 416 to 434 nm
when CdCl2 was added to an aqueous solution of 1a (25 mm in
1:1 50 mm HEPES (pH 7.4)/MeOH), which coincided with a bath-
ochromic absorbance shift with clear isosbestic points (Fig-
ure 2b). The fluorescence and absorbance Job’s plots indicated
which exhibit complexation-in-
duced emission redshifts (Fig-
ure 2c, Table 1). These structural
data suggest that the emission
redshift of the arene–metal-ion
contact is induced by the forma-
tion of a 1:1 binding complex. In
the 1H NMR spectrum of 1a (in
[D6]DMSO, Figure S4 in the Sup-
porting Information) the H(9)
signal is significantly shifted up-
field from 9.31 to 9.00 ppm
upon complexation with CdII,
while the signals of the other
protons on the anthracene ring
remain virtually unchanged. This
result also suggests that the
CdII ion is located at a position
proximate to the C9 carbon of
Figure 2. a) Fluorescence and b) absorbance spectral changes of 1a upon the addition of CdII (0–1.1 equiv).
c) Summary of fluorescence spectral changes of 1a in the presence of 50 mm of metal ions (NiII, CuII, ZnII, AgI, CdII,
HgII, and PbII) and 1 mm of MgII, CaII, SrII, and CsI. Measurement conditions: [1a]=25 mm in 1:1 50 mm HEPES
buffer (pH 7.4)/MeOH, lex =360 nm, 258C. d)–g) X-ray crystallographic analysis of the metal ion complexes: ORTEP
diagrams (50% probability ellipsoids) of d) 1a–CdII (C42H39N7O8Cl2Cd) and f) 1a–AgI (C40H36AgClN6O4). The perchlo-
rate anions and the solvent CH3CN are omitted for clarity. Cross sectional views of e) 1a–CdII and g) 1a–AgI.
the anthracene ring in the solu-
tion state.
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2186
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
showed the emission redshifts upon binding to AgI, CdII, and
HgII (Table 1 and Figures S7 and S10 in the Supporting Informa-
tion). Among the metal complexes of 2a, 2a–AgI complex
showed a smaller redshift than did either 2a–CdII or 2a–HgII
(Table 1). To confirm whether 2a–AgI complex forms the
arene–metal-ion contact, its structure was analyzed by X-ray
crystallographic analysis. The solved structure of 2a–AgI
showed that the distance between the AgI and the closest C9
carbon atom was 2.97 ꢁ, which is less than the sum of the Van
der Waals radii of the AgI ion (1.72 ꢁ) and aromatic carbon
(1.77 ꢁ) (Figure S3 in the Supporting Information). This result
indicates that 2a–AgI forms an arene–metal-ion interaction,
even though this complex showed a small emission redshift
(DF=10 nm).
Table 1. Summary of fluorescent peak maxima shifts (DF [nm]) induced
by metal ions.[a]
Probes (fluorescent peak top [nm])
Metal ions
1a
2a
2b
2c
3a
(Van der Waals
radius [ꢁ])[b]
(416)
(528)
(528.5)
(524.5)
(589)
MgII (1.73)[c]
CaII (2.31)[c]
NiII (1.63)[d]
CuII (1.40)[d]
ZnII (1.39)[d]
SrII (2.49)[c]
AgI (1.72)[d]
CdII (1.58)[d]
CsI (3.43)[c]
HgII (1.55)[d]
PbII (2.02)[d]
0
0
1
0
ꢀ0.5
0
0
0
0
0
–
–
–
0
10
35
0
37.5
0
0
[e]
[e]
[e]
[e]
–
–
–
–
–
–
–
[e]
[e]
[e]
[e]
1.5
0.5
0
8.5
0
0
7.5
0
[e]
[e]
3
1
0
3.5
0
18
0.5
26
11.5
ꢀ1
10
29
0
35.5
0
34.5
18
0
[e]
–
To evaluate the effects of the metal binding sites, further
metal-ion screening was conducted with the other Ida-type
probe 2b and Beta-type probe 2c (Table 1, Figures S8 and S9
in the Supporting Information). The Ida-type probe 2b exhibit-
ed selective emission shifts upon addition of CdII, HgII, and PbII
(Figure S8 in the Supporting Information), while the Beta-type
probe 2c showed emission shifts upon the addition of AgI and
HgII (Figure S9 in the Supporting Information). These results
demonstrate that the arene–metal-ion contact occurs in the
probes with the different metal binding sites. Interestingly, the
DF value changed depending on the type of ligand site em-
ployed. As an example, 2a (DF=29 nm) displayed a larger
emission shift than 2b (DF=18 nm) upon complexation with
CdII. Table 2 summarizes the binding affinities (log Ka) of the
0
[a] Measurement conditions: 1a (25 mm, lex =360 nm); 2a, 2b (5 mm,
lex =500 nm), 2c (10 mm, lex =500 nm); 3a (5 mm, lex =550 nm) in 1:1
50 mm HEPES buffer (pH 7.4)/MeOH. [b] Reported value in the literature
(see reference [6]). [c] The value was measured upon the addition of
100 equiv of metal ion. [d] The value was measured at the saturation
point in the presence of 1–4 equiv of metal ion. [e] Emission shift was not
detectable due to the almost complete fluorescence quenching upon
binding to the metal ion.
Ratiometric metal-ion sensing of xanthene and pyronine-
based probes
The metal-ion sensing was further conducted with 2a and 3a,
which have xanthene and pyronine and fluoresce at long
wavelength above 500 nm. As shown in Figure 3, both 2a and
Table 2. Summary of the binding constant (log Ka) of the probes with
various metal ions.[a]
1a
2a
2b
2c
7.87
<3[c]
3.85
<3[c]
2d
3a
AgI
CdII
HgII
PbII
6.95
7.68
8.50
8.19
7.72
8.89
5.51[c]
7.19
7.59
7.95
5.05
4.97
5.65
6.63
6.26
8.57
[b]
[b]
[b]
[b]
–
–
–
–
[a] Measurement conditions; 50 mm HEPES buffer (pH 7.4)/MeOH (1:1).
[b] Not determined due to the formation of insoluble aggregation. [c] De-
termined by isothermal titration calorimetry (ITC, Figure S19 in the Sup-
porting Information).
Figure 3. Fluorescence spectral changes of a) 2a (lex =500 nm) and b) 3a
(lex =550 nm) upon complexation with CdII (0–1.1 equiv). Measurement con-
ditions: [Probe]=5 mm, 50 mm HEPES buffer (pH 7.4)/MeOH (1:1), 258C.
probes for various metal ions. Intriguingly, 2b did not exhibit
a shift of its fluorescence wavelength (DF=0 nm) upon com-
plexation with AgI (log Ka =5.51), which contrasts with the be-
havior of 2a and 2c (DF=10 and 8.5 nm, respectively) upon
AgI binding. Regarding metal-ion selectivity, 2a preferably
bound to HgII with a strong binding affinity (log Ka =8.89),
while 2b showed a selectivity for PbII (log Ka =7.95) among the
metal ions investigated. Intriguingly, 2c underwent selective
complexation with AgI (log Ka =7.87), the value of which is
more than 1000 times greater than the affinities with the other
metal ions. This high selectivity indicates the potential utility of
the arene–metal contact in development of ratiometric probes
for specific metal ions.
3a exhibit clear ratiometric signal change upon addition of
CdCl2 under aqueous conditions (5 mm of the probe in 1:1
50 mm HEPES (pH 7.4)/MeOH). The emission redshifts (DF) of
2a and 3a induced by CdII were 29 nm and 35 nm (Table 1), re-
spectively, which coincided with the bathochromic absorbance
shifts with clear isosbestic points (Figure S5 in the Supporting
Information). Conversely, the addition of CdII induced no emis-
sion shift in 2d, which has a single Dpa site (Figure S2 in the
Supporting Information). In metal-ion screening, 2a and 3a
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2187
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Theoretical computational analysis of arene–metal-ion
contact
To provide a theoretical basis for understanding the effects of
metal-ion contact on the photophysical properties of the fluo-
rophores, electronic structure calculations were performed for
2a. The calculations indicated a large emission redshift upon
complexation of 2a with metal ions. The low-lying excited
states of 2a and 2a–CdII in water were calculated using the
TDDFT method (Tables S3 and S4 in the Supporting Informa-
tion). The S0!S1 transition with the highest oscillator strength
can be assigned to the first UV/Vis band observed in the spec-
tra, which is attributed to a localized p–p* excitation of the
xanthene moiety. In ligand 2a, the S0!S1 transition energy
was calculated to be 2.90 eV, while the same transition energy
for the 2a–CdII complex was calculated as 2.69 eV, which is
0.21 eV lower than that of 2a. This result reasonably reprodu-
ces the experimentally observed CdII ion-induced redshift of
the absorption band (D of 0.14 eV; 2a 2.44 eV (508 nm)!2a–
CdII 2.30 eV (530 nm)). The S0!S1 transition of 2a–AgI (2.84 eV)
in water was also calculated to be less than that of 2a
(2.90 eV) (Table S5 in the Supporting Information), which
agrees with the observed experimental results of the redshift
upon AgI complexation (D of 0.04 eV; 2a 2.44 eV (508 nm)!
2a–AgI 2.40 eV (516 nm). Further TDDFT calculations were con-
ducted by replacing the CdII ion of 2a–CdII with a positive
charge of +1.0e C (2a–PC; Table S6 in the Supporting Informa-
tion) under vacuum conditions. The S0!S1 transition of 2a–PC
was calculated to be 2.71 eV, a value that is again lower than
that of 2a (2.92 eV) under the same vacuum conditions
(Table S7 in the Supporting Information). In addition, as the
positive charge is increased (0.2e, 0.4e, 0.6e, 0.8e C), the
energy gaps of 2a–PC become lower (data not shown). These
data suggest that the photophysical properties of 2a could be
significantly affected by electrostatic interactions with the
metal ions.
Figure 4. Results of theoretical molecular orbital calculations using TDDFT
method: a) HOMO and LUMO energy diagrams of 2a, 2a–CdII and 2a–AgI in
water as well as 2a and 2a–PC under vacuum and b) shapes and energy
levels of the HOMO and LUMO of 2a, 2a–CdII and 2a–AgI in water. Values in
parentheses indicate the orbital energy levels in hartree units. Color figures
of the molecular orbitals are shown in the Supporting Information.
Figure 4a summarizes the energy levels of the HOMO and
LUMO of 2a and its metal complexes. The data show that the
energy level of the LUMO is more efficiently stabilized by the
metal complexation than that of the HOMO, resulting in
shrinkage of the HOMO–LUMO energy gap. This is consistent
with the observed redshift of the absorption band and the
fluorescence emission upon complexation with the metal ions.
Figure 4b shows the shapes of the HOMO and LUMO orbitals
of 2a, 2a–CdII and 2a–AgI. The data reveal that the C9 carbon
of the xanthene does not contribute to the HOMO, since it is
located at a node of the orbital, while the pp orbital at the C9
position makes a significant contributes to the LUMO. These
topological features of the orbitals explain why the LUMO is
more effectively stabilized by proximate metal ions as com-
pared with the HOMO.
well suited to in cell bioimaging, the probes were attempted
to the ratiometric fluorescence detection of CdII ion in living
cells. Among the various probes, 2a showed the highest bind-
ing affinity and metal-ion selectivity for CdII compared with 2b,
2c, and 3a (Tables 1, 2). This strong binding affinity of 2a for
CdII (log Ka =7.72) enables the selective detection of CdII in the
presence of other metal ions (Figure S11 in the Supporting In-
formation) and glutathione (Figure S17 in the Supporting Infor-
mation). The detection limit (based on 3s) of 2a for CdII was
found to be 13.6 nm in PBS buffer. In addition, no significant
pH-dependent fluorescence ratio changes in either 2a or the
2a–CdII were observed in the physiological pH range from 6 to
8 (Figure S12 in the Supporting Information).
Fluorescence imaging of cadmium(II) ion uptake in living
cell
In CdII imaging inside living cells, the cell-permeable diacety-
lated derivative diAc-2a was prepared (Figure 5a). When HeLa
cells were treated with 5 mm diAc–2a in HBS buffer, bright
fluorescence due to the xanthene was observed throughout
Taking advantage of the large emission redshifts and the long
emission wavelengths (>500 nm) of the probes, which are
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2188
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
that allows to visualize the endocytosis-mediated CdII uptake
in living cells, owing to its high ratiometric fluorescence
change.[4]
Ratiometric fluorescent sensing of hydrogen sulfide in living
cell
It was expected that the present sensing mechanism would be
applicable not only to detection of metal ions, but also to ra-
tiometric sensing of other biological substances that can coor-
dinate to the metal-ion/fluorescent probes complexes. To dem-
onstrate this point, we attempted to ratiometrically monitor
hydrogen sulfide (H2S) in living cells using the CdII complex.
It has recently been revealed that endogenously produced
H2S serves as an important gaseous signal transmitter and me-
diates a number of biological processes, including vasorelaxa-
tion, neurotransmission and inflammation.[9] For this reason,
a number of fluorescent probes have been reported with the
aim of understanding the biological roles of H2S.[10] Most of
these probes, however, are based on H2S-mediated azide re-
duction or trapping of H2S via nucleophilic addition, and suffer
from slow reaction kinetics, which prevents real-time detection
of H2S in biological systems. Recently, Hanaoka et al. have re-
ported the CuII-containing fluorescent probe HSip-1 for H2S de-
tection, which exhibits a rapid, large increase in fluorescence
due to the formation of CuS precipitate to generate a metal-
ion-free fluorescent ligand.[11] Since CdII can form stable and in-
soluble CdS (Ksp of CdS=2ꢃ10ꢀ28 m2) with H2S in aqueous solu-
tion, it was anticipated that our CdII complex would sense H2S
by the same sensing mechanism as HSip-1, with a rapid and
large ratiometric fluorescence signal. We chose the CdII com-
plex of 2a because it showed a larger emission redshift and
a higher stability constant compared to 2b–CdII and 3a–CdII
(Tables 1, 2). In the in vitro titration, 2a–CdII underwent an evi-
dent blueshift in emission in 20 mm HEPES buffer (pH 7.4,
[GSH]=5 mm) upon addition of Na2S. The extent of the shift
depended on the concentration of Na2S, such that the ratio
value R (F527nm/F557nm) changed from 0.42 to 2.37 as the concen-
tration of Na2S was increased up to 20 mm (Figure 6a). ESI-
mass spectrometry confirmed that 2a–CdII released free 2a
upon the addition of Na2S as a result of the formation of CdS
(Figure S16 in the Supporting Information).
The sensing selectivity of 2a-CdII for the biologically impor-
tant thiol species was examined (Figure S17 in the Supporting
Information). Significant changes in the ratio value of 2a–CdII
were not induced by the presence of the physiologically abun-
dant glutathione (GSH, 5 mm), lipoic acid (1 mm) and l-cys-
teine (1 mm). This is a good contrast to 2b-CdII and 3a-CdII,
both of which showed the significant emission shifts upon the
addition of GSH (data not shown). Variations over time in the
value of R induced by Na2S were monitored by real-time fluo-
rescence detection (Figure 6b). In the absence of GSH, the
R values gradually increased and plateaued after 2 min. This re-
sponse was not significantly altered by the presence of 5 mm
GSH, such that the change in R value was complete within
1 min, which is a much faster response than that exhibited by
the CuII-based probe HSip-1 in the presence of GSH.[11] The
Figure 5. Ratiometric fluorescence detection of CdII ion in HeLa cells using
diAc–2a: a) Structure of diAc–2a, b) ratio image of the cells treated with
diAc–2a (5 mm), c) ratio image of the cells after treatment with CdCl2 (5 mm)
and pyrithione (100 mm) for 10 min, d) Ratio image of cells after treatment
with CdCl2 (5 mm) for 5 h. Inset shows the magnified image. Conditions:
[diAc–2a]=5 mm, R=F540–560nm/F510–530nm. Scale bar: 50 mm.
the cells by ratiometric imaging at two detection channels
(F510–530nm; lex =488 nm, lem =510–530 nm and F540–560nm; lex =
514 nm, lem =540–560 nm) (Figure 5b). It was evident that
non-fluorescent diAc–2a penetrated into the cells whereupon
it was hydrolyzed to 2a by intracellular esterases. When cells
pre-stained with diAc–2a were treated with 5 mm CdCl2 and
100 mm pyrithione (PyT), a representative intracellular metal-
ion transporter,[7] the ratio value (R) throughout the cells in-
creased from less than 2 to more than 3 within 10 min (Fig-
ure 5c). In contrast, when HeLa cells were incubated with free
CdCl2 (5 mm) in HBS buffer for 5 h in the absence of PyT, fol-
lowed by staining with diAc–2a, small fluorescent particulates
with the high ratio values (R>3) were clearly observed inside
the cells (Figure 5d). Similar images of fluorescent particulates
were observed in HEK293 cells upon treatment with free CdII
(Figure S13 in the Supporting Information). The image of these
fluorescent particulates overlapped well with that obtained
from Texas Red-conjugated BSA, a representative endosome
marker, but not with that of Lysotrackerꢂ Red DND-99 (Fig-
ure S14 in the Supporting Information). In addition, the incuba-
tion of the cells with CdII at 48C did not produce any traces of
fluorescence particulates (Figure S15 in the Supporting Infor-
mation). These results strongly suggest that free CdII was trans-
ported into the cells via the endosome pathway. CdII has been
recognized as a highly toxic heavy metal capable of causing
a number of lesions and cancers in many organs, although the
mechanisms of CdII ion uptake by and movement within bio-
logical systems remain unclear.[8] To our knowledge, the pres-
ent result represents the first instance of a CdII chemosensor
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2189
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Conclusion
In conclusion, we have developed ratiometric fluorescent
probes for metal ions based on arene–metal-ion contact. We
have clearly demonstrated that various fluorophores and
metal-ion binding units can be employed for design of the
probes, which are useful for ratiometric detection of several
metal ions, such as AgI, CdII, HgII, and PbII. The successful appli-
cations of the probes to in cell ratiometric bioimaging have
also demonstrated the utility of arene–metal-ion contact sens-
ing for bioanalytical applications. An important future project
within our research group will be to further extend this sens-
ing mechanism to other metal ions (such as alkali metals, alkali
earth metals, and 3d metal ions). These metal ions have rather
small ion radii and/or stable hydration structures, both of
which could potentially prevent the interaction between the
metal ion and a fluorophore. However, the theoretical electron-
ic structure calculations predicted that an important factor of
the fluorescence emission shift may be electrostatic interaction
between the metal and the fluorophore, suggesting that
arene–metal-ion contact could be generally applicable to the
sensing of a wider variety of metal ions and biological susbtan-
ces. Further research along these lines is now being performed
by our group.
Experimental Section
Synthesis and characterization of the compounds
Figure 6. Ratiometric fluorescence detection of H2S using 2a–CdII: a) Fluores-
cence emission change of 2a–CdII (10 mm) upon addition of Na2S ([Na2S]=0,
2.5, 5, 6.5, 7.5, 10, 15, and 20 mm) in 20 mm HEPES (pH 7.4), lex =500 nm,
5 mm glutathione (GSH) at 378C. b) Time trace of fluorescence intensity ratio
1a, 2a, 3a, and diAc–1a, were synthesized according to the re-
ported methods.[12] The syntheses and characterizations of
compounds 1d and 2b, 2c, 2d, and Ab–1a are described in
the Supporting Information.
*
R (F527nm/F557nm) upon addition of Na2S in the presence of GSH ( : 5 mm GSH,
&
*
: 30 mm Na2S, : 1 mm GSH+30 mm Na2S, n: 5 mm GSH+30 mm Na2S. Error
bars show standard deviations based on n=3), c) structure of Ab-2a–CdII.
d) Time course plot of the fluorescence ratio change of 2a–CdII in HeLa cells.
Fluorescence measurements
Fluorescence titration with various metal ions were conducted
with a solution (3 mL) of the fluorescence probes in 50 mm HEPES
buffer (pH 7.4)-MeOH (1:1) in a quartz cell (1 cmꢃ1 cm) at 258C.
An aqueous stock solution of metal ion was added to the samples
with a micropipette and the fluorescence emission spectra were re-
corded. Fluorescence titration curves at appropriate wavelengths
were analyzed with the nonlinear least-squares curve-fitting
method to evaluate the apparent binding constant (Ka, mꢀ1). Rela-
tive fluorescence quantum yields (F) of 2a, 2b, 2c, and 2a–CdII
were determined by using a 0.1 N NaOH solution of fluorescein
(F=0.85) as the quantum yield standard. Relative fluorescence
quantum yields (F) of 3a were determined by using a MeOH solu-
tion of Rhodamine B (F=0.65) as the quantum yield standard.
*
*
)
The cells were subjected to fluorescence imaging with ( ) or without (
treatment with Na2S (final conc. 150 mm). Each data point was obtained for
ROIs (n=5) inside cells in three imaging experiments. e, f) Time-lapse ratio
image of HeLa cells (at 0 and 10 min) after treatment with Na2S (final conc.
150 mm). Measurement conditions: [Ab-2a–CdII]=10 mm,
R=F510–530nm/F540–560nm, scale bar: 50 mm.
sensing ability of 2a–CdII for H2S was subsequently examined
in living cells. The trypan blue viability test indicated that nei-
ther the 2a–CdII nor CdII exhibited significant cell toxicity or
cell death induction (Figure S18 in the Supporting Informa-
tion). Since diAc-2a–CdII was membrane impermeable, the
more hydrophobic CdII complex Ab-2a–CdII (Figure 6c) was
prepared. Following the introduction of Ab-2a–CdII (10 mm)
into HeLa cells, the cells were treated with Na2S (150 mm in
final concentration). Time lapse ratiometric imaging showed
that the R value (F510–530nm/F540–560nm) gradually increased from
0.51 to 0.61 over the span of 10 min, while ratiometric change
was not observed without the Na2S addition (Figure 6d). These
results indicate that 2a–CdII effectively detects exogenous H2S
uptake inside cells by a fluorescence ratio change without in-
terfering with the intracellular environment.
X-ray crystallography
1a–CdII (ClO4 as counter anions) was crystallized in distilled
ꢀ
water/CH3CN=1:3 at 308C to obtain a colorless brick crystal.
ꢀ
1a–AgI (ClO4 as counter anions) was crystallized in distilled
water/MeOH=1:3 at 308C to obtain a colorless column crystal.
1a–2ZnII (Clꢀ as counter anions) was crystallized in distilled
water/CH3CN=1:3 at 308C to obtain a colorless brick crystal.
ꢀ
1a–2PbII (NO3 as counter anions) was crystallized in distilled
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2190
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
water/MeOH=1:3 at 308C to obtain a colorless needle crystal.
Acknowledgements
ꢀ
2a–AgI (ClO4 as a counter anion) was crystallized in distilled
water/MeOH=1:3 at 308C to obtain an orange brick crystal.
The X-ray data were collected on a Bruker AXS APEX II diffrac-
tometer with graphite monochromated MoKa radiation (l=
0.71069 ꢁ). The structures were solved by the direct method
and refined anisotropically for non-hydrogen atoms by full-
matrix least-squares calculations. The crystallographic calcula-
tions were performed by using the crystal structure software
package of the Rigaku Corporation.[13] There were some unas-
signed electron densities owing to severely disordered solvent
around special positions. The crystal data and information for
each solved structure is included in Table S2 in the Supporting
Information.
We appreciate the technical supports from the Research Sup-
port Center and Associate Prof. Dr. Karasawa, Graduate School
of Pharmaceutical Sciences, Kyushu University. This work was
supported by Platform for Drug Discovery, Informatics, and
Structural Life Science from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. This work was per-
formed under the Cooperative Research Program of “Network
Joint Research Center for Materials and Devices” organized by
(MEXT, Japan). A.O. acknowledges Toray Science Foundation
for its financial support. I.T. acknowledges the Japan Society
for the Promotion of Science (JSPS) Research Fellowships for
Young Scientists.
Keywords: analytical methods · fluorescent probes · imaging
agents · noncovalent interactions · pi interactions
Theoretical computational analysis
Density functional theory (DFT) was applied to investigate the
electronic origin of the spectral shift induced in the metal com-
plexes. In the calculation, the CAM-B3LYP functional was ap-
plied to describe the exchange-correlation term.[14] This calcula-
tion was carried out for molecules in a vacuum with the de-
fault computational parameters in Gaussian09.[15] In order to
calculate the excited states, the TDDFT method was applied to
obtain the lower 20 states. The Kohn–Sham orbitals were de-
scribed with the Gaussian basis sets. For all the elements
except Cd and Ag, we used the 6–31G(d,p) and the 6–31+
G(d,p) basis sets for optimization.[16] For Cd and Ag, the va-
lence Gaussian basis set for the Stuttgart/Dresden effective
core potential (ECP), represented as SDD,[15] was applied where
the core electrons were replaced with the corresponding ECP.
The solvent effect was taken into account by using the polar-
ized continuum model for water as the solvent.
[1] a) I. Johnson, Histochem. J. 1998, 30, 123–140; b) L. Prodi, M. Montalti,
N. Zaccheroni, L. S. Dolci, Topics in Fluorescence Spectroscopy, Vol. 9,
Springer, New York, 2005, 1–52; c) D. W. Domaille, E. L. Que, C. J.
Chang, Nat. Chem. Biol. 2008, 4, 168–175.
[2] a) S. Kenmoku, Y. Urano, K. Kanda, H. Kojima, K. Kikuchi, T. Nagano, Tet-
rahedron 2004, 60, 11067–11073; b) H. Komatsu, D. Citterio, Y. Suzuki, T.
Kubota, K. Tokuno, Y. Kitamura, K. Oka, K. Suzuki, J. Am. Chem. Soc.
2004, 126, 16353–16360; c) H. He, M. A. Mortellaro, M. J. P. Leiner, S. T.
Young, R. J. Fraatz, J. K. Tusa, Anal. Chem. 2003, 75, 549–555; d) A.
Minta, R. Y. Tsein, J. Biol. Chem. 1989, 264, 19449–19457; e) G. Grynkie-
wicz, M. Poenie, R. Y. Tsien, J. Biol. Chem. 1985, 260, 3440–3450.
[3] a) X. Chen, T. Pradhan, F. Wang, J. S. Kim, J. Yoon, Chem. Rev. 2012, 112,
1910–1956; b) M. Taki, J. L. Wolford, T. V. O’Halloran, J. Am. Chem. Soc.
2004, 126, 712–713; c) S. C. Burdette, G. K. Walkup, B. Spingler, R. Y.
Tsien, S. J. Lippard, J. Am. Chem. Soc. 2001, 123, 7831–7841.
[4] a) T. Cheng, Y. Xu, S. Zhang, W. Zhu, X. Qian, L. Duan, J. Am. Chem. Soc.
2008, 130, 16160–16161; b) M. Taki, M. Desaki, A. Ojida, S. Iyoshi, T. Hir-
ayama, I. Hamachi, Y. Yamamoto, J. Am. Chem. Soc. 2008, 130, 12564–
12565; c) W. Liu, L. Xu, R. Sheng, P. Wang, H. Li, S. Wu, Org. Lett. 2007, 9,
3829–3832.
[5] a) F. Fages, J. P. Desvergne, H. B. Laurent, P. Marsau, J. M. Lehn, F. K.
Hibert, A. M. Albrecht-Gary, M. A. Joubbehll, J. Am. Chem. Soc. 1989, 111,
8672–8680; b) M. E. Huston, C. Engleman, A. W. Czarnick, J. Am. Chem.
Soc. 1990, 112, 7054–7056; c) M. Choi, M. Kim, K. D. Lee, K.-N. Han, I.-A.
Yoon, H.-J. Cung, J. Yoon, Org. Lett. 2001, 3, 3455–3457; d) Y. Hitomi, T.
Nagai, M. Kodera, Chem. Commun. 2012, 48, 10392–10394.
[6] a) M. Mantina, A. C. Chamberlin, R. Valero, C. J. Cramer, D. G. Truhlar, J.
Phys. Chem. A 2009, 113, 5806–5812; b) A. Bondi, J. Phys. Chem. 1964,
68, 441–451.
Fluorescence imaging of CdII ion uptake in living cells
All fluorescence imaging was conducted with a confocal laser scan-
ning microscope (A1Rsi, Nikon) equipped with a 60ꢃ objective
lens. The following detection channels were chosen for the ratio-
metric imaging; F510-530nm: lex =488 nm, lem =510–530 nm, and
F540–560nm: lex =514 nm, lem =540–560 nm. For the imaging of CdII-
pyrithione (PyT) uptake, HeLa cells were incubated with diAc–2a
(5 mm) in HBS buffer (107 mm NaCl, 6 mm KCl, 1.2 mm MgSO4,
2 mm CaCl2, 11.5 mm glucose, 20 mm HEPES, pH 7.4) for 20 min at
378C. After washing twice with HBS buffer, the cells were treated
with CdCl2 (5 mm) and PyT (100 mm) at 378C for 30 min, prior to ra-
tiometric imaging. For the imaging of free CdII uptake (without
PyT), HeLa cells were incubated with CdCl2 (5 mm) in the modified
HBS buffer (without CaCl2 and MgSO4) for 5 h at 378C. After wash-
ing twice with HBS buffer, the cells were treated with diAc–2a
(5 mm) at 378C for 20 min, prior to ratiometric imaging.
[7] K. Komatsu, K. Kikuchi, H. Kojima, Y. Urano, T. Nagano, J. Am. Chem. Soc.
2005, 127, 10197–10204.
[8] a) F. Thꢄvenod, Biometals 2010, 23, 857–875; b) E. V. Kerkhove, V. Pen-
nemans, Q. Swernen, Biometals 2010, 23, 823–855; c) D. A. Vesey, Toxi-
col. Lett. 2010, 198, 13–19; d) H. Fujishiro, S. Okugaki, K. Kubota, T. Fu-
jiyama, H. Miyataka, S. Himeno, J. Appl. Toxicol. 2009, 29, 367–373; e) H.
Gunshin, B. Mackenzie, U. V. Berger, Y. Gunshin, M. F. Romero, W. F.
Boron, S. Nussberger, J. L. Gollan, M. A. Hedinger, Nature 1997, 388,
482–488.
[9] a) L. Li, A. Hsu, P. K. Moore, Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169–
187; b) L. Li, A. Hsu, P. K. Moore, Pharmacol. Ther. 2009, 123, 386–400;
c) C. Szabꢅ, Nat. Rev. Drug Discovery 2007, 6, 917–993.
[10] a) V. S. Lin, C. J. Chang, Curr. Opin. Chem. Biol. 2012, 16, 595–601;
b) V. S. Lin, A. R. Lippert, C. J. Chang, Proc. Natl. Acad. Sci. USA 2013, 110,
7131–7135; c) S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E. H. Joe, B. R. Cho,
H. M. Kim, J. Am. Chem. Soc. 2013, 135, 9915–9923; d) F. Yu, P. Li, P.
Song, B. Wang, J. Zhao, K. A. Han, Chem. Commun. 2012, 48, 2852–
2854; e) Y. Qian, L. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J.
Zhao, C. He, Nat. Commun. 2011, 2, 495–501.
Ratiometric imaging of hydrogen sulfide in HeLa cells
HeLa cells were incubated with a solution of Ab-2a (10 mm) and
CdCl2 (20 mm) for 20 min at 378C in the modified HBS buffer (with-
out CaCl2 and MgSO4). After washing twice with HBS buffer, the
cells were treated with sodium sulfide (150 mm final conc.) and
then subjected to ratiometric imaging.
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2191
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[11] K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T.
Ueno, T. Terai, H. Kimura, T. Nagano, J. Am. Chem. Soc. 2011, 133,
18003–18005.
[12] a) Y. Kurishita, T. Kohira, A. Ojida, I. Hamachi, J. Am. Chem. Soc. 2012,
134, 18779–18789; b) A. Ojida, I. Takashima, T. Kohira, H. Nonaka, I. Ha-
machi, J. Am. Chem. Soc. 2008, 130, 12095–12101; c) A. Ojida, Y. Mito-
oka, K. Sada, I. Hamachi, J. Am. Chem. Soc. 2004, 126, 2454–2463; d) A.
Ojida, Y. Mito-oka, M. Inoue, I. Hamachi, J. Am. Chem. Soc. 2002, 124,
6256–6258.
bayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyen-
gar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B.
Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,
S. Dapprich, A. D. Daniels, ꢆ. Farkas, J. B. Foresman, J. V. Ortiz, J. Cio-
slowski, D. J. Fox, Gaussian, Inc. Wallingford CT, 2009.
[16] The geometry optimizations were carried out for complexes whose
crystallographic data are available. The calculated distance between AgI
and C9 in 2a–AgI was 2.93 and 3.17 ꢁ in vacuum and in water, respec-
tively, while the experimental value is 2.97 ꢁ. In the case of 1a–CdII, the
calculated CdII–C9 distance is 3.22 and 3.67 ꢁ in vacuum and in water,
respectively, while the experimental value is 3.14 ꢁ. These results indi-
cate that the optimized geometries in vacuum are much closer to the
experimentally determined structure compared to that in water. There-
fore, we adopted the calculated in vacuo structure for the analysis.
[13] CrystalStructure 3.8.2: Crystal Structure Analysis Package, Rigaku and
Rigaku/MSC (2000–2003). 9009 New Trails Dr. The Woodlands TX
77381USA.
[14] T. Yanai, D. P. Tew, N. C. Handy, Chem. Phys. Lett. 2004, 393, 51–57.
[15] Gaussian 09, Revision A.02, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men-
nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian,
A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro,
M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Ko-
Received: October 26, 2013
Published online on January 23, 2014
Chem. Eur. J. 2014, 20, 2184 – 2192
www.chemeurj.org
2192
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim