Ratiometric fluorescent sensor based on inhibition of resonance for detection of cadmium in aqueous solution and living cells

Article abstract of DOI:10.1021/ic200032e

Although cadmium has been recognized as a highly toxic heavy metal and poses many detrimental effects on human health, the Cd2⁺-uptake and nosogenesis mechanisms are still insufficiently understood, mainly because of the lack of facile analytical methods for monitoring changes in the environmental and intracellular Cd2⁺ concentrations with high spatial and temporal reliability. To this end, we present the design, synthesis, and photophysical properties of a cadmium sensor, DQCd1 based on the fluorophore 4-isobutoxy-6-(dimethylamino)-8-methoxyquinaldine (model compound 1). Preliminary investigations indicate that 1 could be protonated under neutral media and yield a resonance process over the quinoline fluorophore. Upon excitation at 405 nm, 1 shows a strong fluorescence emission at 554 nm with a quantum yield of 0.17. Similarly, DQCd1 bears properties comparable to its precursor. It exhibits fluorescence emission at 558 nm (Φf = 0.15) originating from the monocationic species under physiological conditions. Coordination with Cd2⁺ causes quenching of the emission at 558 nm and simultaneously yields a significant hypsochromic shift of the emission maximum to 495 nm (Φf = 0.11) due to inhibition of the resonance process. Thus, a single-excitation, dual-emission ratiometric measurement with a large blue shift in emission (Δλ = 63 nm) and remarkable changes in the ratio (F_{495 nm}495/F_{558 nm}) of the emission intensity (R/R ₀ up to 15-fold) is established. Moreover, the sensor DQCd1 exhibits very high sensitivity for Cd2⁺ (K_d = 41 pM) and excellent selectivity response for Cd2⁺ over other heavy- and transitionmetal ions and Na⁺, K⁺, Mg2⁺, and Ca2⁺ at the millimolar level. Therefore, DQCd1 can act as a ratiometric fluorescent sensor for Cd2⁺ through inhibition of the resonance process. Confocal microscopy and cytotoxicity experiments indicate that DQCd1 is cellpermeable and noncytotoxic under our experimental conditions. It can indeed visualize the changes of intracellular Cd2⁺ in living cells using dual-emission ratiometry.

Full text of DOI:10.1021/ic200032e

ARTICLE
pubs.acs.org/IC
Ratiometric Fluorescent Sensor Based on Inhibition of Resonance
for Detection of Cadmium in Aqueous Solution and Living Cells
Lin Xue,† Guoping Li,†,‡ Qing Liu,†,‡ Huanhuan Wang,†,‡ Chun Liu,§ Xunlei Ding,† Shenggui He,*,† and
Hua Jiang*,†
†Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190,
People's Republic of China
§State Key Laboratory of Fine Chemicals, Department of Chemical Engineering, Dalian University of Technology, Dalian 116012,
People's Republic of China
‡Graduate School of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
S Supporting Information
b
ABSTRACT: Although cadmium has been recognized as a highly toxic heavy metal and poses many detrimental effects on human
health, the Cd2þ-uptake and nosogenesis mechanisms are still insufficiently understood, mainly because of the lack of facile
analytical methods for monitoring changes in the environmental and intracellular Cd2þ concentrations with high spatial and
temporal reliability. To this end, we present the design, synthesis, and photophysical properties of a cadmium sensor, DQCd1 based
on the fluorophore 4-isobutoxy-6-(dimethylamino)-8-methoxyquinaldine (model compound 1). Preliminary investigations
indicate that 1 could be protonated under neutral media and yield a resonance process over the quinoline fluorophore. Upon
excitation at 405 nm, 1 shows a strong fluorescence emission at 554 nm with a quantum yield of 0.17. Similarly, DQCd1 bears
properties comparable to its precursor. It exhibits fluorescence emission at 558 nm (Φf = 0.15) originating from the monocationic
species under physiological conditions. Coordination with Cd2þ causes quenching of the emission at 558 nm and simultaneously
yields a significant hypsochromic shift of the emission maximum to 495 nm (Φf = 0.11) due to inhibition of the resonance process.
Thus, a single-excitation, dual-emission ratiometric measurement with a large blue shift in emission (Δλ = 63 nm) and remarkable
changes in the ratio (F495 nm/F558 nm) of the emission intensity (R/R0 up to 15-fold) is established. Moreover, the sensor DQCd1
exhibits very high sensitivity for Cd2þ (Kd = 41 pM) and excellent selectivity response for Cd2þ over other heavy- and transition-
metal ions and Naþ, Kþ, Mg2þ, and Ca2þ at the millimolar level. Therefore, DQCd1 can act as a ratiometric fluorescent sensor for
Cd2þ through inhibition of the resonance process. Confocal microscopy and cytotoxicity experiments indicate that DQCd1 is cell-
permeable and noncytotoxic under our experimental conditions. It can indeed visualize the changes of intracellular Cd2þ in living
cells using dual-emission ratiometry.
’ INTRODUCTION
of their simplicity, high sensitivity, and high resolution of
fluoroscopy.7 Historically, some fluorescent sensors for Cd2þ
have been reported,8 but only a few of them are applicable to
cellular imaging.9 Moreover, most of these sensors are intensity-
based and do not provide sufficient accuracy for quantitative
measurements because their emission intensity is known to be
conditioned by many variables, such as the sample environment,
sensor concentration, bleaching, and instrumental efficiency. In
contrast, ratiometric sensors can eliminate most or all ambigu-
ities by built-in calibration of the two emission bands.10 To this
end, several sensors have been developed and utilized in ratio-
metric Cd2þ imaging in living cells.11 In spite of their fascinating
responses to Cd2þ, these sensors still have some hangups, such as
poor water solubility, UV excitation, and large spectral overlap,
and should be refined further. To date, it is still a tremendous
challenge to design Cd2þ-selective sensors, in particular, ratio-
metric sensors for the accurate detection of Cd2þ in aqueous
solutions and biological environments.
Cadmium (Cd) and its derivatives are extremely toxic, and
their bioaccumulations, consequently, result in many serious
diseases and even many types of cancers.1 The EPA (United States
Environmental Protection Agency) gives an enforceable drinking
water standard for Cd of 5 ppb to prevent kidney damage and
other related diseases, while the WHO (World Health
Organization) provides a more strict guideline value for Cd of 3
ppb for drinking water.2 Recent reports revealed that the multiple
cytotoxic and metabolic effects of Cd may principally originate
from interference with the normal functions of essential metals
such as Zn2þ and Ca2þ 3,4
Besides, Cd is also involved in inducing
.
oxidative stress and altering the activities of various enzymes.5
Although these delectable progresses in the disclosure of toxic
effects of Cd have been achieved, the Cd2þ-uptake and nosogen-
esis mechanisms are still poorly understood.6 Thus, it is highly
desirabletodevelopreliableanalyticaltechniques for detecting and
monitoring Cd in drinking water, environmental samples, and
cell/tissues so as to clarify the biological roles of Cd.
Recently, fluorescent sensors capable of optically sensing
transition-metal ions have attracted increasing attention because
Received: January 6, 2011
Published: March 11, 2011
r
2011 American Chemical Society
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Scheme 1. Resonance Process of the Protonated 4-Methox-
yquinoline (Pyridine) Derivatives
Scheme 2. Design Strategy for 1 and DQCd1
It is well-known that an essential resonance effect exists in
protonated 4-alkoxy-substituted quinoline (pyridine) derivatives
(Scheme 1).12 The resonance charge transfer from the electron-
donating methoxy group to the quinolinic (pyridinic) nitrogen would
cause the charge to delocalize, resulting in a resonant oxonium
electronic structure. Consequently, this resonance would greatly
stabilize the conjugate acid and lead to a drastic red shift in the
absorption wavelength. We speculate that a dye bearing such a feature
can be utilized in the design of a sensor in which the signal readout is
regulated by switching of the resonance process upon protonation or
deprotonation. Therefore, we designed the model compound 1
(Scheme 2), which possesses the intramolecular charge-transfer
pathway from the dimethylamino group to the quinolinic nitrogen,
and expected that the quinolinic nitrogen would be protonated under
neutral aqueous solutions because of the strengthened basicity
originating from the presence of 6-dimethylamino and 4-isobutoxy
groups.12,13 So, a similar resonance would take place and stabilize the
protonated species, consequently leading to long-wavelength absorp-
tion and fluorescence emission.
On the other hand, the di-2-picolylamine (DPA) moiety was
initially employed for Zn2þ-selective sensors and has been widely
investigated so far.14 In fact, most of these Zn2þ sensors exhibit similar
significant responses to Cd2þ as well because of the close chemical
properties between Zn2þ and Cd2þ. However, when incorporated
with auxiliary receptors, the DPA moiety is also available for the design
of Cd2þ-selective sensors with preserved superiorities such as a high
affinity for Cd2þ and fast Cd complex formation, high selectivity for
Cd2þ over Ca2þ, Mg2þ, Naþ, and Kþ, and good membrane
permeability.11,15 When such a chelator, which would create a
metal-ion binding pocket with participation of the quinolinic nitrogen
atom, is incorporated into model compound 1 to obtain the sensor
DQCd1, the resonance process of the protonated DQCd1 can be
turned off by coordination-induced deprotonation of DQCd1. Con-
sequently, inhibition of resonance and charge delocalization causes a
distinct blue-shift fluorescence emission and provides a single-excita-
tion, dual-emission ratiometric measurement for metal cations.
reaction failed, direct O-alkylation using 1-iodo-2-methylpro-
pane under basic conditions produced model compound 1 in
moderate yield. Then the hydrochloric salt of 1 was obtained by
acidification of 1 with HCl (1 M). Compound 1 was further
oxidized to generate aldehyde 8, which was combined with DPA
in dry 1,2-dichloroethane in the presence of NaB(OAc)3H to
give DQCd1 in reasonable yield after column chromatography
and recrystallization. Finally, the metal complexes of DQCd1
were obtained through the reaction of DQCd1 with Zn(NO3)2
and CdCl2. Suitable single crystals for X-ray crystallography were
obtained by solvent diffusion.
Resonance in 1 and DQCd1. We first studied the photo-
physical properties of model compound 1. The fluorescence
spectra show that emission of 1 remains constant in a wide pH
range from 4 to 7 and is quenched gradually as the pH reaches 9.
Clearly, two protonation/deprotonation events affect the fluor-
escence emission. According to the similar protonation/depro-
tonation steps of 6-(dimethylamino)quinaldine (6-DQ)13 with
pKa1 = 6.8 (ring nitrogen) and pKa2 = 1.9 (exocyclic nitrogen),
the apparent pKa values of 7.67 and 2.46 (Figure 1) for 1 can be
assigned to the nitrogen atoms at the quinolinic ring and amino
group, respectively. On the other hand, the pKa values of 7.61 and
2.41 obtained from UVꢀvis measurements are very similar to
those based on fluorometric experiments (Figure S3 in the
Supporting Information). These pKa values indicate that pro-
tonation of 1 takes place under neutral aqueous media in the
ground state. It should be noted that in contrast to the pKa values
of 6-DQ, compound 1 shows a stronger tendency to form
monoprotonated species at neutral media. This basicity-strength-
ening effect could be explained as the existence of resonance over
the quinolinic fluorophore. Such a resonance process was further
confirmed by theoretical calculations. Density functional theory
(DFT) computations reveal that the positive charge of the
monoprotonated species distributes over the isobutoxy group
and quinolinic nitrogen. Further, 1H NMR studies also demon-
strate that the CH2 signal of the isobutoxy proton in 1 undergoes
a remarkable downfield shift (Δδ = 0.29 ppm) upon protonation
(see NMR section), clearly indicating the presence of a partial
positive charge on the isobutoxy group and the existence of
resonance. In addition, the DFT calculations also suggest that the
energy of the S0 f S1 transition of the protonated species is
much lower than that of neutral species (Supporting In-
formation), consistent with the large red shift in the experimental
absorption wavelength (∼400 nm). Upon excitation at 405 nm,
1 shows a strong fluorescence emission at 554 nm with a
quantum yield of 0.17 in aqueous solutions (pH = 7.4).
’ RESULTS AND DISCUSSION
Synthesis. The synthetic strategies for preparing 1, 1 HCl,
3
and DQCd1 are outlined in Scheme 3. The starting compound
was initially protected with acetic anhydride in 92% yield to
generate 2. The reduction of the nitro group in 2 to an amino
group by hydrogenation using Pd/C was efficient and afforded
the quantitative product of 3. Compound 4 was prepared by
alkylation of 3 with formaldehyde and sodium borohydride in
acidic media (yield 89%)16 and further deprotected to achieve
the o-anisidine precursor 5. The enamine 6, which was prepared
by condensation of the o-anisidine 5 with ethyl acetoacetate, was
cyclized thermally (reflux of diphenyl oxide) under nitrogen to
give quinolone 7 (yield of about 71%). Although an attempt to
alkylate the 4-position oxygen of 7 through the Mitsunobu
Next, we were encouraged to investigate the effect of pH on
UVꢀvis absorption and fluorescence emission of DQCd1
(Figures 1 and S4 in the Supporting Information). Although
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Scheme 3. Synthetic Strategy for 1, 1 HCl, and DQCd1a
3
a Reagents and conditions: (a) Ac2O, pyridine, 120 °C, 92%. (b) H2, Pd/C, EA, rt, >95%. (c) 40% aqueous HCHO, NaBH4, THF/H2SO4, 0 °C, 89%.
(d) H2SO4, MeOH, 60 °C, >95%. (e) Ethyl acetoacetate, AcOH, benzene, reflux, 60%. (f) Diphenyl oxide, 250 °C, 71%. (g) 1-Iodo-2-methylpropane,
K2CO3, DMF, 80 °C, 60%. (h) SeO2, dioxane, 80 °C, 57%. (i) DPA, NaBH(AcO)3, rt, 90%. (j) EtOH, HCl (1.0 M), rt, 91%. (k) CdCl2 or Zn(NO3)2,
MeOH, rt.
hydrogen bonding as acceptors for the proton so that the nearby
positive charge originating from the proton binding pocket
results in a smaller pKa (4.46) of the pyridine moiety in DQCd1
than that of free pyridine (5.17).18 This similar pocketlike
binding environment for the proton was also proposed for
fluorescein-DPA-based sensors by Lippard et al.17 These findings
demonstrate that DQCd1 can be protonated under physiological
conditions and displays a resonance process similar to that of
model compound 1.
Optical Responses of DQCd1 to Cd2þ. With these data on
hand, we chose a 10 mM HEPES, 0.1 M NaCl, pH = 7.4 aqueous
Figure 1. (a) Normalized emission intensity of 1 (0, λem = 554 nm)
and DQCd1 (O, λem = 558 nm) in aqueous buffer (10 mM HEPES and
0.1 M NaCl); λex = 405 nm. The solid lines represent the nonlinear least-
buffer for investigating the abilities of DQCd1 to sense Cd2þ
(Table 1). In this solution, DQCd1 showed a good solubility and
stable photophysical properties even after 25 days (Figure S5 and
S6 in the Supporting Information). The absorption spectra of
free DQCd1 showed a clear charge-transfer band around 420 nm
(ε = 1.9 ꢁ 103 Mꢀ1 cmꢀ1), resulting from its monoprotonated
species. Upon the addition of Cd2þ, this band decreased
gradually, accompanied by a new blue-shift band around
370 nm with five distinct isosbestic points at 388, 348, 335,
311, and 277 nm (Figure 2a), implying a different coordination
process between quinoline fluorophore and Cd2þ from its
analogues.15b,19 The saturated spectra were readily obtained
when 1 equiv of Cd2þ was introduced, suggesting a strong
affinity for Cd2þ and the formation of a 1:1 complex
(Figure 2a, inset), which was further confirmed by Job’s plots,
1H NMR titration experiments, and X-ray crystallographic
analysis (Figures 3, 6, and S7 in the Supporting Information).
The free ligand shows a strong fluorescence emission at 558 nm
with a quantum yield Φf of 0.15 upon excitation at 405 nm.
Titration of DQCd1 with Cd2þ caused quenching of the
emission at 558 nm. Simultaneously, a new blue-shift emission
band appeared at 495 nm with a large hypsochromic shift of
63 nm and a distinct isoemission point at 528 nm (Figure 2b).
squares fits to the experimental data.
the DPA-based DQCd1 bears multiple potential protonation
sites, indeed, we only found two protonation/deprotonation
processes from our photometric experiments, which have appar-
ent pKa values of 7.31 and 4.46 (7.45 and 4.65) from the
fluorescence (UVꢀvis) spectra. According to the model com-
pound 1, the larger pKa was assigned to the quinolinic nitrogen,
but the smaller one was assigned to one of 2-pyridyl arms rather
than the dimethylamino nitrogen.17 The pKa1 (7.31) of DQCd1
is about 0.4 unit lower in comparison with that (7.67) of 1,
suggesting a weaker basic site of the quinolinic nitrogen. We
speculated that the quinolinic nitrogen, the tertiary nitrogen,
2-pyridyl groups of DPA, and the methoxy group constituted a
binding pocket that was occupied by a proton (Scheme 4). This
implies that the quinolinic nitrogen is not the sole site of
protonation and the tertiary amine and pyridine groups are also
involved in protonation, which, in turn, inhibits the photoin-
duced electron-transfer quenching effect of tertiary amine. Thus,
DQCd1 exhibits relatively high fluorescence emission (Φf =
0.15). In addition, the 2-pyridyl groups also participate in
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Scheme 4. Chemical Equilibria of DQCd1 To Form Protonated and Metal-Bound Complexes
Table 1. Spectroscopic Data for the Compoundsa
compound
λabsb [nm]
ε ꢁ 104 [Mꢀ1 cmꢀ1
]
λemb [nm]
Φfc
1
252
258
262
263
2.88
3.74
4.80
4.34
554
558
495
510
0.17
0.15
0.11
0.06
DQCd1
Cd-DQCd1
Zn-DQCd1
a 10 mM HEPES, 0.1 M NaCl, pH = 7.4, 25 °C. b The maximum
absorption or emission intensity. c Quinine sulfate was used as the
standard for quantum yield measurements.
The quantum yield of DQCd1 was found to be 0.11 in the
presence of 1 equiv of Cd2þ. The ratios of the fluorescence
intensities at 495 and 558 nm were found to be 0.2 and 3.1 in the
absence and presence of Cd2þ, respectively, indicating that
DQCd1 is an excellent dual-emission ratiometric fluorescent
sensor for Cd2þ (R/R0 up to 15). These results also demonstrate
that the proton at the quinolinic site of DQCd1 was displaced by
Cd2þ because of strong coordination between Cd2þ and
DQCd1, consequently resulting in inhibition of resonance and
affording a ratiometric approach to the detection of Cd2þ
(Scheme 4). Thus, DQCd1 exhibits a large blue shift in emission
and a remarkable increase in the intensity ratio (F495 nm/F558 nm
)
upon binding to Cd2þ and provides a good opportunity to
generate high-resolution images for cellular Cd2þ using dual-
emission ratiometry.
Figure 2. (a) Absorption spectra of 20 μM DQCd1 upon titration of
Cd2þ (0ꢀ40 μM) in aqueous buffer (10 mM HEPES, 0.1 M NaCl, pH =
7.4). Inset: Absorbance changes as a function of the Cd2þ concentration.
(b) Fluorescence spectra (λex = 405 nm) of 10 μM DQCd1 upon
1H NMR Studies. The protonation and metal-ion coordina-
tion events were further investigated by 1H NMR data in
deuterated dimethyl sufloxide (DMSO-d6; Figure 3), and assign-
titration of Cd2þ (0ꢀ20 μM) in aqueous buffer. Inset: Ratio (F495 nm
/
F
558 nm) changes as a function of the Cd2þ concentration.
1
1
oxygen atom, substantially confirming the existence of reso-
nance. Next, we examined the metal binding of DQCd1
(Figure 3b). The remarkable proton shifts of the DPA moiety
and the 3-position proton H(10) indicate that Cd2þ coordinated
to the DPA moiety and the quinoline fluorophore. Interestingly,
the chemical shift of the proton H(10) displayed an upfield shift,
and the isobutoxy H(60) showed a slight downfield shift upon the
addition of Cd2þ. These results indicate that charge distribution
of the butoxy oxygen is less affected by Cd2þ coordination to the
quinolinic nitrogen atom, and the resonance effect does not take
ments of the proton signals are based on Hꢀ H COSY and
HMBC experiments (see the Supporting Information). We first
1
obtained H NMR spectra of 1 and 1 HCl. As shown in
3
Figure 3a, protons H(10) and H(90) display remarkable down-
field shifts up to Δδ = 0.59 and 0.29 ppm upon protonation,
respectively, hinting at the existence of a positive charge at the
quinoline nitrogen. Notably, the chemical shift of the proton
H(60) of the isobutoxy group also underwent a significant
downfield shift from 3.95 to 4.24 ppm (Δδ = 0.29 ppm), which
clearly shows delocalization of the partial positive charge on the
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Figure 3. Partial 1H NMR spectra (400 MHz) of (a) 1 (5 mM) and 1 HCl (5 mM). (b) DQCd1 (5 mM) and DQCd1 þ Cd2þ (5 mM) in DMSO-d6.
3
Figure 4. (a) Fluorescence spectra of DQCd1 (10 μM) in the presence of various metal ions (10 μM Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ
,
Hg2þ, and Pb2þ and 1 mM Mg2þ, Ca2þ, and Kþ) in a buffer solution (10 mM HEPES, 0.1 M NaCl, pH = 7.4, λex = 405 nm). (b) Visible emission
observed from samples of DQCd1 (L), Cd-DQCd1, and Zn-DQCd1, λex = 365 nm. (c) Metal-ion selectivity profiles of DQCd1 (10 μM) in the absence
(top) and presence (bottom) of 50 μM NTA. Gray bars represent the fluorescence intensity ratio responses (F495 nm/F558 nm) of DQCd1 in the
presence of various metal ions. Black bars represent the ratio responses of DQCd1 in the presence of the indicated metal ions, followed by 10 μM Cd2þ
.
place because of the strong electron-withdrawing effect of the
metal ion.
has little interference on Cd2þ sensing. In fact, in some more
complicated systems, such as seawater and biological samples,
the practicability for detection of Cd2þ may be diminished by the
high level of other 3d metal ions, especially for Zn2þ. To further
eliminate this interference, we chose nitrilotriacetic acid (NTA)
as a masking agent because NTA has higher affinities for Zn2þ
(log KZn = 10.66) than those for Cd2þ (log KCd = 9.78).20 So,
selectivity experiments were repeated in the presence of 5 equiv
of NTA under otherwise identical conditions. Although Zn2þ
Metal-Ion Selectivity. The selectivity profiles of DQCd1
were examined by titrations with various potential competing
metal ions (Figure 4). Naþ, Kþ, Ca2þ, and Mg2þ, which are
abundant in water and living samples, exerted a negligible effect
on the fluorescence response for Cd2þ even at high concentra-
tions (1 mM). Co2þ, Ni2þ, and Cu2þ strongly quenched the
fluorescence (Figures 4a and S8a in the Supporting In-
formation), even in the presence of an equal equivalent of
Cd2þ. Zn2þ partially quenched the emission of DQCd1 (ΦZn
= 0.055) but with a new emission at 510 nm and a smaller
hypsochromic shift (Δλ = 48 nm) than that of Cd2þ. These
results indicate that DQCd1 is able to distinguish Cd2þ from
Zn2þ by both fluorescence emission wavelength and intensity
upon metal binding, even by “naked eyes” (Figure 4b). Adding
Cd2þ to a Zn-DQCd1 solution caused a rapid increase in both
the fluorescence intensity and the ratio signals within 10 min
(Figure S8 in the Supporting Information), implying that Zn2þ
and other transition-metal ions (Mn2þ, Fe2þ, Co2þ, Ni2þ, Cu2þ
,
Hg2þ, and Pb2þ) result in the partial quenching of fluorescence
but without a distinct blue shift (Figure S9 in the Supporting
Information), the ratio values of F495 nm to F558 nm (Figure 4c,
bottom, gray bars) are much smaller than those in the absence of
NTA (Figure 4c, top, gray bars). As expected, only Cd2þ induced
an obvious blue shift of the emission and a significant increase in
the ratio. The R/R0 value of 13.5 is comparable with that in the
absence of NTA (R/R0 = 15). Moreover, the Cd2þ-induced ratio
value (F495 nm/F558 nm) was not affected by the presence of the
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Table 2. Selected Bond Distances (Å) for the Crystal
Structuresa
compound
Cd-DQCd1
Zn-DQCd1
MꢀN1
MꢀN2
MꢀN3
MꢀN4
MꢀO1
MꢀX
2.304(4)
2.457(4)
2.352(5)
2.278(5)
2.647(4)
2.4472(17)
2.0688(16)
2.2089(16)
2.0993(16)
2.1444(15)
2.4939(14)
2.0587(15)
a M represents the metal ion Cd(2) or Zn(1); X represents the solvent
molecule coordinated to the metal ion.
Figure 5. Ratio fluorescence responses (F495 nm/F558 nm) of DQCd1 as
a function of free Cd2þ (O) or Zn2þ (0) concentrations (M = Cd2þ or
Zn2þ). The solid lines represent nonlinear least-squares fits to the
experimental data.
above-mentioned metal ions (Figure 4c, bottom, black bars).
Further experiments showed that, even in the media with an
NTA concentration of 0.5 mM, 0.1 mM Zn2þ did not seriously
interfere with the response (R/R0 ≈ 10) of DQCd1 to Cd2þ at
the micromolar level (Figure S10 in the Supporting Informa-
tion). These data indicate that NTA can eliminate unfavorable
effects of other transition metals, in particular Zn2þ, on the
response of DQCd1 to Cd2þ. Moreover, the related ratio value
(R = F495 nm/F558 nm) of DQCd1 and Cd-DQCd1 showed a pH
independence in the range of 6ꢀ8 (Figure S12 in the Supporting
Information). Thus, DQCd1 is suitable for monitoring intracel-
lular Cd2þ based on the ratiometric assay under physiological
conditions.
Figure 6. Crystal structures of the complexes (a) Cd-DQCd1 and (b)
Zn-DQCd1. Thermal ellipsoids are shown at the 50% probability level.
All hydrogen atoms and counteranions are omitted for clarity.
Binding Affinities. The accession of fluorescent molecules
with high binding affinity is very important in the development of
Cd2þ-selective sensors.8h,11b,11c It was shown that the Cd2þ
concentrations are usually 1 ppb or even lower for unpolluted
natural waters,21,22 which are elevated by industrial discharge or
contamination of municipal wastewater, and certain bioaccumu-
lations in some shellfish take place in Cd2þ-contaminated sea-
water with a concentration as low as 10ꢀ7 M.22 On the other
hand, lower Cd2þ concentrations down to 100 pM were also
found to significantly induce cell growth and DNA synthesis.23
So, fluorescent sensors with dissociation constants (Kd values) in
the subnanomolar or picomolar range will be practical to Cd2þ
detection in these pools.
partially quenched by nonradiative deactivations via vibronic
coupling with the vibrational states of OꢀH oscillators.8f Accord-
ingly, Cd2þ and Zn2þ induced significantly different fluorescence
responses in aqueous solution but comparable fluorescence
responses in an aqueous acetonitrile solution (HEPES buffer with
50% CH3CN; Figure S15 in the Supporting Information). Similar
phenomena were also described by us and Liu et al.8f,15b
The limit of detection (LOD) of DQCd1 for Cd2þ (Figure
S13 in the Supporting Information) was further determined to be
9.6 pM (1.1 ng/L), which is far lower than the maximum limit for
drinking water emphasized by the WHO and EPA. In contrast
with the traditional approach for the detection of Cd2þ in the
environment, such as anodic stripping voltammetry (LOD of 1
ng/L)24 and atomic absorption spectrometry (LOD of 5 ng/
L),25 we believe that DQCd1 affords sufficient sensitivity for the
detection of a trace amount of Cd2þ in environmental samples.
X-ray Crystallographic Analysis. The solid states of metal
complexes of DQCd1 were investigated by X-ray crystallogra-
phy. Initially, we attempted to crystallize various metal salts
The dissociation constant of DQCd1 for Cd2þ was deter-
mined by fluorescence spectroscopy in metalꢀligand-buffered
solutions with different Cd2þ concentrations. DQCd1 re-
sponded to the picomolar concentration of free Cd2þ, and Kd
was determined by using nonlinear least-squares fit analysis of the
ratio of the fluorescence intensity (F495 nm/F558 nm; Figure 5) to
be 4.1 ( 0.3 ꢁ 10ꢀ11 M (41 pM), indicating the high binding
affinity of DQCd1 for Cd2þ. The Kd value of DQCd1 for Zn2þ
was also calculated to be 1.3 ( 0.1 ꢁ 10ꢀ9 M, which is similar
with that of the previously reported sensor HQ1.19 These
findings indicate that the 8-position methoxy group is propitious
to binding Cd2þ, as observed in other 8-methoxyquinoline-based
sensors.8b,d,f,15b,19 Thus, we infer that the larger Cd2þ (ionic radii
rþ = 0.96 Å for Cd2þ and rþ = 0.74 Å for Zn2þ) may be more
suitable for the coordination cavity in space, resulting in a
stronger interaction between Cd2þ and the methoxy oxygen
and a consequent high affinity. On the other hand, the zinc
complex lacking a suitable ion radius may be easily disrupted by
protic solvent molecules, and fluorescence of this complex is
[MCl2, MSO4, M(ClO4)2, M(NO3)2, where M = Cd2þ or Zn2þ
]
with DQCd1 in a variety of solvents. Indeed, the crystals suitable
for crystallographic analysis were obtained in methanol/ethyl
ether26 and methanol/ethyl ether/H2O27 for Cd-DQCd1 and
Zn-DQCd1, respectively. The crystal structures and data of both
complexes are shown in Figure 6, Table 2, and the Supporting
Information. In the Cd complex Cd-DQCd1, each asymmetric
unit contains two similar Cd coordination sets, where Cd2þ is
chelated by quinoline, a chloride anion, and the DPA moiety to
form a disordered trigonal-bipyramidal coordination geometry.
Meanwhile, the distance between methoxy O1 and Cd2 of
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Figure 8. Fluorescence ratio images of Cd2þ in HEK 293 cells labeled
with 10 μM DQCd1 in PBS buffer. (a) Bright-field image. (b) Ratio
image of emission window F430ꢀ490 nm/F530ꢀ590 nm. (cꢀh) Time-lapse
ratio imaging of the cells after treatment with 30 μM CdCl2 over 1 h. (i
and j) Ratio images after treatment with 50 μM TPEN for 5 and 10 min.
530ꢀ590 nm decreased, whereas the intensity of the emission
collected at 430ꢀ490 nm increased (Figure 7, middle). Accord-
ingly, significant enhancement in the ratio of F430ꢀ490 nm to
F530ꢀ590 nm can be obtained (Figure 7d). These are consistent
with the observations in aqueous buffer and demonstrate that
DQCd1 can readily reveal variation of the intracellular Cd2þ
concentrations (Figure 7, middle). Moreover, variations in
fluorescence could be reversed by the subsequent addition of a
50 μM membrane-permeable chelator N,N,N0,N0-tetrakis(2-pyr-
idylmethyl)ethylenediamine (TPEN) into the medium, indicat-
ing that an increase in the ratio of the emission originating from
the response of DQCd1 to intracellular Cd2þ (Figure 7,
bottom). We further carried out confocal experiments in HEK
293 cells with DQCd1. As shown in Figures 8 and S17 in the
Supporting Information, the ratio of F430ꢀ490 nm to F530ꢀ590 nm
gradually increased after treatment of CdCl2 and remained
almost unchanged after 40 min. Similarly, the changes in the
ratios were recovered by subsequent TPEN treatment. The data
obtained from two cell lines demonstrate that DQCd1 is able to
visualize the explicit alteration of intracellular Cd2þ through the
ratio images, which is obviously superior to intensity-based
images of the sole emission channel.
Figure 7. Confocal fluorescence images of intracellular Cd2þ in NIH
3T3 cells with 10 μM DQCd1 in a PBS buffer: NIH 3T3 cells incubated
with DQCd1 (10 μM) at 37 °C for 30 min (top); DQCd1-stained cells
exposed to 30 μM CdCl2 at 25 °C for 25 min (middle); sequestration of
intracellular Cd2þ by the addition of 50 μM TPEN (bottom). (a) Bright-
field images. (b) Fluorescence images with emission collected at
430ꢀ490 nm. (c) Fluorescence images with emission collected at
530ꢀ590 nm. (d) Ratio images generated from parts b and c,
F
430ꢀ490 nm/F530ꢀ590 nm.
2.647(4) Å is obviously longer than that of a typical CdꢀO bond,
indicative of a weak coordination effect. Zn2þ adopts a similar
coordination geometry in Zn-DQCd1, except that a nitrate
anion instead of a chloride anion is coordinated to Zn2þ. The
methoxy oxygen also undergoes weak interaction to Zn2þ with a
Zn1 O1 distance of 2.494(4) Å, as is observed in its analogues
3 3 3
in which the distance can reach as long as 2.688(4) Å.19 However,
the MꢀN coordination bonds fit the typical bond ranges in both
complexes.28 These data demonstrate that the DPA moiety plays
the main function of grasping the metal ions, while the 8-position
methoxy oxygen can be used to tune the selectivity of the sensor.
Ratiometric Fluorescence Imaging of Cd2þ in Living Cells.
The cytotoxicity of the sensor is very essential with respect to the
potential studies on the toxicity effect of Cd2þ. So, we measured
the viability of the DQCd1-treated NIH 3T3 cells by the CCK-8
assay. The cytotoxicity measurements demonstrate that DQCd1
is nontoxic up to 10 μM over at least a 24-h period (Table S5 in
the Supporting Information). To further evaluate whether the
sensing properties of DQCd1 toward Cd2þ in vitro are preserved
in intracellular conditions, we chose two different cell lines, NIH
3T3 (murine fibroblast cell line, a fibroblast cell) and HEK293
(human embryonic kidney cell line, an epithelial cell) to deter-
mine exogenous Cd2þ in living cells by using fluorescence
microscopy.
’ CONCLUSION
We have investigated the absorption and fluorescence emis-
sion properties of two quinoline derivatives, 1 and DQCd1, and
find that 1 and DQCd1 with 4-isobutoxy groups can be proto-
nated in aqueous solutions and thus generate the resonance
process. We further demonstrated our strategy on the develop-
ment of the fluorescent ratiometric sensor, DQCd1, by utilizing
cation-induced inhibition of the resonance process. Such a sensor
provides a single-excitation, dual-emission ratiometric detection
of Cd2þ with a significant blue shift in emission and remarkable
changes in the ratio (F495 nm/F558 nm) of the emission intensity.
Moreover, the cell-permeable and noncytotoxic DQCd1 can
indeed visualize the changes of intracellular Cd2þ in living cells. It
can thus be predicted that the design strategy of cation-induced
inhibition of the resonance process allows one to design new
ratiometric sensors for other metal ions of biological interest.
NIH 3T3 cells, which were incubated with 10 μM DQCd1 for
30 min in a phosphate-buffered saline (PBS) buffer at 37 °C and
washed, showed clear intracellular fluorescence at an emission
channel collected at 530ꢀ590 nm, indicating that DQCd1 is cell-
permeable (Figure 7). Costaining experiments with the com-
mercial acidotropic dye LysoTracker Red (1 μM) confirm that
DQCd1 located at the acidic compartments of the cells shows
the stronger fluorescence (Figure S16 in the Supporting In-
formation). When the cells were treated with 30 μM CdCl2 at
25 °C for 25 min, the intensity of the emission collected at
’ EXPERIMENTAL SECTION
Materials and Methods. Unless otherwise noted, materials were
obtained from Alfa Aesar and were used without further purification. Di-
2-picolylamine (DPA) was purchased from J&K Chemical Ltd. Lyso-
Tracker Red DND-99 was purchased from Invitrogen. All solvents were
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purified and dried by standard methods prior to use. Pure water (18.2
Ω) was used to prepare all aqueous solutions. Melting points were
measured using an X-4 melting point apparatus with a microscope. 1H
and 13C NMR spectra were recorded using a Bruker AVANCE-400 400
MHz spectrometer. Two-dimensional NMR experiments were taken on
a Bruker AVANCE-600 (600 MHz) spectrometer. Chemical shifts are
expressed in parts per million (δ) using residual solvent protons as
internal standards. IR data were recorded on a Bruker Tensor-27
spectrometer. Mass spectra (electrospray ionization, ESI) and high-
resolution mass spectra (ESI) were obtained on LC-MS 2010 and
Bruker Apex IV Fourier transform mass spectrometers, respectively.
Elemental analyses were performed on a Vario EL analyzer (Elementar
Analysensysteme GmbH). UVꢀvis absorption spectra were obtained
using a Hitachi 3010 UVꢀvis spectrometer. Fluorescence spectra were
obtained using a Hitachi F-4600 spectrometer.
was slowly added. The mixture was heated at 60 °C and was stirred for 4
h. The reaction mixture was cooled and neutralized with sodium
hydroxide, and then CH2Cl2 (15 mL ꢁ 5) was added. The organic
solutions were combined, washed with water and brine, and dried with
Na2SO4. Compound 5 was obtained as a purple oil and used without
further purification. TLC: Rf = 0.30 (silica, 25:1 CH2Cl2/MeOH). 1H
NMR (400 MHz, CDCl3, ppm): δ 6.67 (d, J = 8.40 Hz, 1H), 6.40 (s,
1H), 6.30 (d, J = 6.40 Hz, 1H), 3.85 (s, 3H), 3.35 (br, 2H), 2.84 (s, 6H).
13C NMR (100 MHz, CDCl3, ppm): δ 148.4, 145.4, 127.9, 116.2, 106.7,
99.5, 55.5, 42.3. MS (ESI): m/z 167.1 [M þ Hþ].
Synthesis of Compound 6. A solution of compound 5 (1.0 g, 6
mmol), ethyl acetoacetate (1.6 g, 12 mmol), and AcOH (0.5 mL) in
benzene (30 mL) was refluxed for 8 h, removing water with a
DeanꢀStark apparatus. This mixture was cooled to room temperature
and concentrated in vacuum. The residue was purified by flash chroma-
tography (silica gel, petroleum ether/0ꢀ10% ethyl acetate) to give the
desired products as a white solid (1.0 g, 60% based on compound 4).
TLC: Rf = 0.40 (silica, 5:1 petroleum ether/ethyl acetate). Mp:
Synthesis of Compound 2. A total of 10 g of 2-methoxy-4-
nitroaniline and 2 equiv of acetic anhydride (11.5 mL) in 35 mL of
pyridine was heated in a round-bottomed flask (100 mL) for 2 h at
120 °C. The solvent was evaporated under vacuum and azeotroped with
toluene (twice) to generate a crude solid, which was purified by
recrystallization from methanol to give the desired products as a yellow
crystalline residue (11.5 g, 92%). TLC: Rf = 0.48 (silica, 1:1 petroleum
1
109ꢀ110 °C. H NMR (400 MHz, CDCl3, ppm): δ 9.86 (s, 1H),
6.96 (d, J = 8.40 Hz, 1H), 6.27 (br, 2H), 4.63 (s, 1H), 4.17 (q, J = 7.08
Hz, 2H), 3.83 (s, 3H), 2.96 (s, 6H), 1.85 (s, 3H), 1.28 (t, J = 7.08 Hz,
3H). 13C NMR (100 MHz, CDCl3, ppm): δ 170.6, 161.4, 154.9, 150.1,
127.6, 117.9, 104.1, 96.7, 83.9, 58.5, 55.7, 40.9, 20.0, 14.8. FTIR
(KBr, cmꢀ1): 3283, 2982, 2900, 2813, 1652, 1599, 1566, 1523, 1492,
1442, 1387, 1362, 1262, 1161, 1060, 1034, 978, 805, 782, 659, 543. MS
(ESI): m/z 279.2 [M þ Hþ]. ESI-HRMS. Calcd for [M þ Hþ]
C15H23N2O3: m/z 279.1709. Found: m/z 279.1697. Calcd for [M þ
Naþ] C15H22N2NaO3: m/z 301.1528. Found: 305.1518. Calcd for [M
þ Kþ] C15H22N2KO3: 317.1267. Found: 317.1258.
1
ether/ethyl acetate). Mp: 157ꢀ158 °C. H NMR (400 MHz, CDCl3,
ppm): δ 8.58 (d, J = 8.92 Hz, 1H), 7.96 (br s, 1H), 7.93 (d, J = 9.0 Hz,
1H), 7.75 (s, 1H), 4.00 (s, 3H), 2.26 (s, 3H). 13C NMR (100 MHz,
CDCl3, ppm): δ 168.7, 147.2, 142.9, 133.9, 118.2, 117.6, 105.1, 56.3,
25.0. FTIR (KBr, cmꢀ1): 3281, 1675, 1590, 1510, 1338, 1284, 1096,
1027, 880, 801. MS (ESI): m/z 211.1 [M þ Hþ], 233.1 [M þ Naþ].
ESI-HRMS. Calcd for [M þ Hþ] C9H11N2O4: m/z 211.0719. Found:
m/z 211.0706. Calcd for [M þ Naþ] C9H10N2NaO4: m/z 233.0538.
Found: m/z 233.0526.
Synthesis of Compound 7. A mixture of compound 6 (1.00 g, 3.6
mmol) and diphenyl ether (15.0 g) was heated with stirring at 250 °C for
60 min under nitrogen. This mixture was cooled, and the product began
to precipitate in the reaction medium. After cooling, the mixture was
diluted with 100 mL of petroleum ether to complete the precipitation.
The solid was filtered off, washed with ethyl acetate, and recrystallized
from methanol to give of the product as a yellow crystalline residue (0.59
g, 71%). Mp: 272ꢀ273 °C. TLC: Rf = 0.37 (silica, 1:1 petroleum ether/
isopropyl alcohol). 1H NMR (400 MHz, DMSO-d6, ppm): δ 10.78 (s,
1H), 6.77 (s, 2H), 5.78 (s, 1H), 3.98 (s, 3H), 2.94 (s, 6H), 2.32 (s, 3H).
13C NMR (100 MHz, MeOD, ppm): δ 179.2, 150.6, 149.9, 149.1, 127.0,
125.0, 108.7, 101.0, 96.5, 56.4, 41.0, 19.6. FTIR (KBr, cmꢀ1): 3055,
2937, 2893, 1629, 1592, 1556, 1505, 1438, 1395, 1372, 1263, 1229, 1203,
1147, 1076, 997, 881, 845, 820, 762, 549, 532, 499. MS (ESI): m/z 233.2
[M þ Hþ]. ESI-HRMS. Calcd for [M þ Hþ] C13H17N2O2: m/z
233.1290. Found: m/z 233.1279.
Synthesis of Compound 3. A solution of compound 2 (1.06 g, 5
mmol) in dry ethyl acetate (30 mL) was stirred at room temperature
with 0.1 g of 5% Pd/C. After 8 h, the mixture was filtered off and the
crude product was filtered on Celite and rinsed with ethyl acetate.
Compound 3 was obtained as a purple oil and used without further
purification. TLC: Rf = 0.21 (silica, 4:1 CH2Cl2/ethyl acetate). 1H NMR
(400 MHz, CDCl3, ppm): δ 8.04 (d, J = 8.36 Hz, 1H), 7.45 (br, 1H),
6.28ꢀ6.25 (m, 2H), 3.81 (s, 3H), 2.15 (s, 3H). 13C NMR (100 MHz,
CDCl3, ppm): δ 167.8, 149.3, 143.3, 121.7, 119.6, 107.1, 98.5, 55.7, 24.8.
MS (ESI): m/z 181.1 [M þ Hþ], 203.1 [M þ Naþ].
Synthesis of Compound 4. A slurry of compound 3 (0.9 g, 5
mmol) and finely crushed sodium borohydride (1.9 g, 50 mmol) in
tetrahydrofuran (THF; 8 mL) was added to a stirred mixture of 3 M
H2SO4 (2 mL) and 40% aqueous formaldehyde in an Erlenmeyer flask at
a rate compatible with temperature control (0 ( 5 °C). After the
addition was complete, the mixture was made strongly basic with solid
sodium hydroxide and was extracted with ethyl acetate (20 mL ꢁ 3).
The organic solutions were combined, washed with water and brine, and
dried with Na2SO4. The solvents were evaporated to give the crude
product, which was purified by flash chromatography (silica gel,
CH2Cl2/5ꢀ10% ethyl acetate) to give the desired products as a white
solid (0.93 g, 89% based on compound 2). TLC: Rf = 0.5 (silica, 4:1
CH2Cl2/ethyl acetate). Mp: 113ꢀ114 °C. 1H NMR (400 MHz, CDCl3,
ppm): δ 8.11 (d, J = 8.76 Hz, 1H), 7.45 (br s, 1H), 6.33ꢀ6.3 (m, 2H),
3.87 (s, 3H), 2.92 (s, 6H), 2.16 (s, 3H). 13C NMR (100 MHz, CDCl3,
ppm): δ 167.7, 149.3, 148.1, 121.4, 118.1, 104.8, 96.3, 55.5, 41.0, 24.6.
FTIR (KBr, cmꢀ1): 3273, 2994, 2888, 2809, 1647, 1534, 1484, 1420,
1362, 1323, 1130, 1031, 981, 805, 780, 602, 518, 459. MS (ESI): m/z
209.1 [M þ Hþ], 231.1 [M þ Naþ]. ESI-HRMS. Calcd for [M þ Hþ]
C11H17N2O2: m/z 209.1290. Found: m/z 209.1278. Calcd for [M þ
Naþ] C11H16N2NaO2: m/z 231.1109. Found: m/z 231.1097.
Synthesis of Compound 1. A mixture of compound 7 (233 mg, 1
mmol), 1-iodo-2-methylpropane (220 mg, 1.2 mmol), and K2CO3 (0.55
g, 4 mmol) in N,N-dimethylformamide (DMF; 8 mL) was heated at
80 °C for 8 h. After cooling, H2O (30 mL) was added to the mixture and
extracted with ethyl acetate (10 mL ꢁ 3). The organic solutions were
combined, washed with water and brine, and dried with Na2SO4. The
solvents were evaporated to give the crude product, which was purified
by flash chromatography (silica gel, CH2Cl2/0ꢀ30% acetone) to give
the desired products as a pale-yellow solid (184 mg, 60%). TLC: Rf =
1
0.38 (silica, 25:2 CH2Cl2/MeOH). Mp: 135ꢀ136 °C. H NMR (400
MHz, CDCl3, ppm): δ 6.81 (s, 1H), 6.67 (s, 1H), 6.56 (s, 1H), 4.04 (s,
3H), 3.92 (d, J = 6.40 Hz, 2H), 3.05 (s, 6H), 2.67 (s, 3H), 2.26 (m, 1H),
1.12 (d, J = 6.56 Hz, 6H). 13C NMR (100 MHz, CDCl3, ppm): δ 160.5,
155.3, 155.0, 148.2, 135.0, 121.9, 102.0, 98.9, 92.7, 74.4, 55.9, 41.2, 28.3,
25.9, 19.5. FTIR (KBr, cmꢀ1): 2959, 2926, 2870, 2808, 1622, 1597,
1504, 1463, 1415, 1388, 1357, 1260, 1219, 1150, 1087, 998, 820, 777,
671, 645, 575. MS (ESI): m/z 289.2 [M þ Hþ]. ESI-HRMS. Calcd for
[M þ Hþ] C17H25N2O2: m/z 289.1916. Found: m/z 289.1909. Anal.
Synthesis of Compound 5. Compound 4 (1.2 g, 6 mmol) was
dissolved in 20 mL of MeOH. Then 1 mL of concentrated sulfuric acid
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Calcd for C17H26N2O3 (1 H2O): C, 66.64; H, 8.55; N, 9.14. Found: C,
66.56; H, 8.47; N, 9.24.
(s, 1H), 6.65 (s, 1H), 4.28 (s, 3H), 4.08ꢀ4.02 (m, 8H), 3.04 (s, 6H),
2.19 (m, 1H), 1.06 (d, J = 6.6 Hz, 6H). FTIR (KBr, cmꢀ1): 2961, 2930,
2875, 2806, 1623, 1591, 1510, 1466, 1439, 1417, 1387, 1349, 1267, 1223,
1151, 1125, 1075, 1016, 997, 907, 827, 768, 640, 544, 483. ESI-HRMS.
Calcd for [DQCd1 þ Clꢀ þ Cd2þ] C29H35CdClN5O2: m/z 634.1513.
Found: m/z 634.1501.
Synthesis of Zn-DQCd1. DQCd1 (8 mg, 0.016 mmol) was
dissolved in 1 mL of methanol. To this solution was added an aqueous
Zn(NO3)2 solution (90 μL, 0.2 M) at room temperature. The mixture
was shaken for 5 min and placed in a glass tube. Ethyl ether was then
added carefully to the tube. After several days, colorless crystals of the
zinc complex appeared and were ready for X-ray diffraction. 1H NMR
(400 MHz, DMSO-d6, ppm): δ 8.59 (d, J = 5.16 Hz, 2H), 8.1 (t, J = 7.6
Hz, 2H), 7.66 (d, J = 7.88 Hz, 2H), 7.61 (t, J = 6.32 Hz, 2H), 7.0 (s, 1H),
6.93 (s, 1H), 6.59 (s, 1H), 4.52 (q, J = 16.44 Hz, 4H), 4.30 (s, 2H), 4.27
(s, 3H), 4.01 (d, J = 6.36 Hz, 2H), 3.06 (s, 6H), 2.15 (m, 1H), 1.02 (d, J =
6.6 Hz, 6H). FTIR (KBr, cmꢀ1): 3486, 2962, 2930, 1614, 1590, 1512,
1469, 1385, 1303, 1224, 1153, 1076, 1023, 994, 912, 803, 769, 648, 501.
ESI-HRMS. Calcd for [DQCd1 þ NO3ꢀ þ Zn2þ] C29H35N6O5Zn: m/
z 611.1960. Found: m/z 611.1957.
Quantum Yield Measurements. The quantum yield for fluor-
escence was obtained by a comparison of the integrated area of the
corrected emission spectrum of the samples with that of a solution of
quinine sulfate in 0.1 N H2SO4 (Φ = 0.54).29 The concentration of the
reference was adjusted to match the absorbance of the test sample.
Quantum yields of the metal-free ligands were measured in a HEPES
buffer (10 mM HEPES, 0.1 M NaCl, pH = 7.4) containing 25 μM
ethylenediaminetetraacetic acid (EDTA). Quantum yields of metal-
bound ligands were measured in the HEPES buffer containing 10 μM
Cd(ClO4)2 or Zn(ClO4)2. The concentration of the reference was
adjusted to match the absorbance of the test sample at the wavelength of
excitation. Emission for each compound was integrated from 400 to
650 nm with excitation at 360 nm. The quantum yields were calculated
with the expression in eq 1.
3
Synthesis of Compound 1 HCl. A mixture of compound 1 (50
3
mg, 0.16 mmol), HCl (1 M, 0.16 mL), and EtOH (5 mL) was stirred at
room temperature for 10 min. The solvents were evaporated to give a
crude product, which was purified by recrystallization from ethyl
acetate/CH2Cl2 to give the desired products as a yellow solid (54 mg,
1
91%). Mp: 182ꢀ183 °C. H NMR (400 MHz, DMSO-d6, ppm): δ
14.14 (s, 1H), 7.35 (s, 1H), 7.07 (s, 1H), 6.64 (s, 1H), 4.24 (d, J = 6.36
Hz, 2H), 4.14 (s, 3H), 3.10 (s, 6H), 2.78 (s, 3H), 2.24 (m, 1H), 1.09 (d,
J = 6.56 Hz, 6H). FTIR (KBr, cmꢀ1): 2962, 2877, 2812, 1613, 1494, 1445,
1419, 1355, 1326, 1270, 1233, 1150, 1087, 989, 906, 878, 831, 809, 757,
635, 584. ESI-HRMS. Calcdfor C17H25N2O2: m/z 289.1916. Found:m/z
289.1904. Anal. Calcd for C17H30N2O4.5Cl (1 HCl 2.5H2O): C, 55.20;
3
3
H, 8.18; N, 7.57. Found: C, 55.30; H, 8.07; N, 7.68.
Synthesis of Compound 8. A solution of compound 1 (107 mg,
0.35 mmol) in dioxane (20 mL) was heated to 60 °C. To this solution
was added SeO2 (78 mg, 0.7 mmol). The temperature was then
increased to 80 °C. After 2.5 h, the mixture was cooled to ambient
temperature. The precipitate was filtered off. The solvents were evapo-
rated to give the crude product, which was purified by flash chroma-
tography (silica gel, petroleum ether/0ꢀ30% ethyl acetate) to give the
desired products as a yellow solid (60 mg, 57%). TLC: Rf = 0.56 (silica,
1:1 petroleum ether/ethyl acetate). Mp: 194ꢀ195 °C. 1H NMR (400
MHz, CDCl3, ppm): δ 10.14 (s, 1H), 7.31 (s, 1H), 6.79 (d, J = 1.76 Hz,
1H), 6.68 (d, J = 1.64 Hz, 1H), 4.12 (s, 3H), 4.02 (d, J = 6.48 Hz, 2H),
3.16 (s, 6H), 2.28 (m, 1H), 1.13 (d, J = 6.68 Hz, 6H). 13C NMR (100
MHz, CDCl3, ppm): δ 194.1, 160.2, 156.8, 150.6, 148.9, 134.7, 126.1,
98.1, 97.3, 91.6, 74.9, 56.2, 40.6, 28.2, 19.4. FTIR (KBr, cmꢀ1): 2967,
2917, 2807, 1618, 1600, 1499, 1471, 1438, 1415, 1379, 1309, 1260, 1243,
1211, 1158, 1124, 1068, 1000, 887, 822, 782, 723, 634, 588, 430. MS
(ESI): m/z 303.2 [M þ Hþ], 325.2 [M þ Naþ]. ESI-HRMS. Calcd for
[M þ Hþ] C17H23N2O3: m/z 303.1709. Found: m/z 303.1700. Calcd
for [M þ Naþ] C17H22N2NaO3: m/z 325.1528. Found: m/z 325.1519.
Synthesis of DQCd1. To a solution of compound 8 (150 mg, 0.5
mmol) and DPA (0.1 g, 0.5 mmol) in 1,2-dichloroethane (10 mL) was
added NaBH(OAc)3 (0.13 g, 0.6 mmol) in portions. The resulting
solution was stirred at room temperature overnight. Then the solvents
were evaporated, and the solid was diluted with ethyl acetate, washed
with water and brine, and dried over Na2SO4. The solvents were
evaporated to give the crude product, which was purified by flash
chromatography (silica gel, CH2Cl2/0ꢀ5% methanol) to give the
desired products as a yellow solid (0.223 mg, 90%). TLC: Rf = 0.40
(silica, 15:1 CH2Cl2/MeOH). Mp: 101ꢀ102 °C. 1H NMR (400 MHz,
CDCl3, ppm): δ 8.54 (d, J = 4.6 Hz, 2H), 7.65ꢀ7.57 (m, 4H), 7.28 (s,
1H), 7.13 (t, J = 5.72 Hz, 2H), 6.79 (d, J = 2.08 Hz, 1H), 6.66 (d, J = 1.4
Hz, 1H), 4.05ꢀ4.03 (m, 5H), 3.97 (d, J = 6.4 Hz, 2H), 3.93 (s, 4H), 3.05
(s, 6H), 2.26 (m, 1H), 1.13 (d, J = 6.68 Hz, 6H). 13C NMR (100 MHz,
CDCl3, ppm): δ 160.9, 159.6, 156.3, 155.4, 149.2, 148.4, 136.4, 123.2,
122.8, 122.0, 100.8, 99.0, 92.5, 74.6, 61.1, 60.2, 56.0, 41.1, 28.2, 19.5.
FTIR (KBr, cmꢀ1): 3051, 3011, 2959, 2929, 2872, 2815, 1621, 1589,
1503, 1469, 1435, 1360, 1258, 1218, 1176, 1144, 1087, 1047, 1024, 980,
880, 820, 765, 635. MS (ESI): m/z 486.4 [M þ Hþ], 508.3 [M þ Naþ].
ESI-HRMS. Calcd for [M þ Hþ] C29H36N5O2: m/z 486.2869. Found:
R
emissionsample
R
Φsample ¼ Φstandard
ð1Þ
emissionstandard
Determination of Protonation Constants. The apparent pKa
was measured by plotting the emission intensity and UVꢀvis absorption
at selected wavelengths against the pH recorded in the range from ∼2 to
∼11. A solution of 1 and DQCd1 containing 10 mM HEPES and 0.1 M
NaCl was acidified by adding HCl (6 N), and the UVꢀvis (20 μM) and
fluorescence spectra (10 μM) were recorded. Aliquots of 6, 3, 1, 0.5, and
0.1 N NaOH were added to achieve the appropriate pH changes (ΔpH =
0.3ꢀ0.5), and the UVꢀvis and fluorescence spectra were recorded after
each addition. The overall volume change for each experiment did not
exceed ∼2%. Throughout the titration, the temperature was maintained
at 25 ( 0.5 °C by circulating constant-temperature water through the
water jacket of the titration cell. The resulting absorption (A) or
normalized fluorescence emission intensity (F) was plotted as a function
of the pH and fitted to the expression in eq 230 to calculate the pKa
values. ΔY1 and ΔY2 are the maximum absorption (A) or fluorescence
emission intensity (F) changes associated with the corresponding pKa
values.
m/z 486.2865. Anal. Calcd for C29H36N5O2.5 (DQCd1 0.5H2O): C,
3
70.42; H, 7.34; N, 14.16. Found: C, 70.41; H, 7.38; N, 14.04.
Synthesis of Cd-DQCd1. DQCd1 (8 mg, 0.016 mmol) was
ΔY1
1 þ 10pH ꢀ pK
ΔY2
1 þ 10pH ꢀ pK
ΔY ¼
þ
ð2Þ
dissolved in 1 mL of methanol. To this solution was added CdCl2 2.5
a1
a2
3
H2O (4 mg, 0.018 mmol) at room temperature. The mixture was shaken
for 5 min and placed in a glass tube. Ethyl ether was then added carefully
to the tube. After several days, colorless crystals of the Cd complex
Determination of Dissociation Constants. Fluorescence in-
tensity ratio values of 10 μM DQCd1 as a function of the free metal-ion
concentration were measured in a HEPES buffer solution. Free Cd2þ
concentrations were obtained by using a 1 mM EDTA, 5 mM
1
appeared and were ready for X-ray diffraction. H NMR (400 MHz,
DMSO-d6, ppm): δ 9.0 (d, J = 4.6 Hz, 2H), 7.95 (t, J = 7.12 Hz, 2H),
7.54 (t, J = 6.24 Hz, 2H), 7.48 (d, J = 7.8 Hz, 2H), 7.0 (s, 1H), 6.93
3688
dx.doi.org/10.1021/ic200032e |Inorg. Chem. 2011, 50, 3680–3690

Inorganic Chemistry
ARTICLE
Mg(NO3)2, and 0.05ꢀ0.9 mM Cd(ClO4)2 buffer system. Free Zn2þ
concentrations were obtained by using a 2 mM NTA and 0.2ꢀ1.9 mM
Zn(ClO4)2 buffer system (see the Supporting Information). The
solutions were allowed to equilibrate at 25 ( 0.5 °C for 5 min after
each addition. The ratio value (F495 nm/F558 nm) was plotted and fitted to
eqs 3 and 4 as described.31
For investigation of the intracellular distribution of DQCd1, 5 μM
DQCd1 and 1 μM Lysotracker Red DND-99 in the culture media
containing 0.2% (v/v) DMSO were added to the cells, and the cells were
incubated for 30 min at 37 °C. After washing with PBS (5 mL ꢁ 3) to
remove the excess sensor, the cells were then filled with 2 mL of PBS for
fluorescence imaging. The cells were imaged with a FV1000 confocal
laser-scanning microscope equipped with the appropriate excitation and
emission filters for DQCd1 (λex = 405 nm; λem = 425ꢀ475 nm) and
Lysotracker Red (λex = 543 nm; λem = 555ꢀ655 nm).
½M2þꢂfreeRmax þ KdξRmin
Rðλ1em=λ2emÞ ¼
ð3Þ
ð4Þ
2þ
Kdξ þ ½M
ꢂ
free
For ratiometric imaging of intracellular Cd2þ, 10 μM DQCd1 in the
culture media containing 0.2% (v/v) DMSO was added to the cells, and
the cells were incubated for 30 min at 37 °C. After washing with PBS
(5 mL ꢁ 3) to remove the excess sensor, the cells were treated with 30
μM CdCl2 in PBS for 5ꢀ60 min. Fluorescence at two emission channels
of 430ꢀ490 and 530ꢀ590 nm was measured at room temperature by
excitation at 405 nm. The ratio images of F430ꢀ490 nm/F530ꢀ590 nm were
obtained on a pixel-by-pixel basis using FV10-ASW version 0107A-1
software. Subsequent treatment with TPEN (50 μM in the media) was
performed directly on the microscope stage. Images were then collected
and processed again with the Olympus FV1000 system.
Fminðλ2emÞ
Fmaxðλ2emÞ
ξ ¼
Metal-Ion Selectivity. The fluorescence spectra of 2 mL aliquots
of 10 μM DQCd1 were acquired in a HEPES buffer solution after the
addition of an aliquot of metal stock solutions [the final metal’s
concentrations are 1.0 mM for KNO3, Mg(NO3)2, or Ca(NO3)2 and
10 μM for MnCl2, FeSO4 (newly prepared), Co(NO3)2, NiSO4,
Cu(NO3)2, Zn(ClO4)2, HgCl2, or Pb(NO3)2]. After acquisition, an
aliquot of Cd(ClO4)2 (10 μM) was further titrated into the relevant
solutions, and the fluorescence values of competing samples were
measured again. Excitation was provided at 405 nm, and the emission
intensity ratio R was calculated as F495 nm/F558 nm. The same experi-
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures, charac-
b
terization, and additional fluorescence and UVꢀvis absorption
spectra of 1, 1 HCl, and DQCd1, H and 13C NMR, Hꢀ H
1
1
1
mental procedures were repeated in the presence of 50 and 500 μM
3
2ꢀ
NTA, respectively. The effects of counteranions such as NO3ꢀ, SO4
,
COSY, and HMBC spectra, and X-ray crystallographic data of
Zn-DQCd1 and Cd-DQCd1 in CIF format. This material is
available free of charge via the Internet at http://pubs.acs.org.
CO32ꢀ, SO32ꢀ, ClO4ꢀ, and AcOꢀ on the emission of DQCd1 were also
examined, and no interference was observed (Figure S11 in the
Supporting Information).
X-ray Crystallographic Analysis. Measurements were done at
113.2 K on a Rigaku RAXIS-RAPID imaging-plate diffractometer
equipped with a CCD detector using Mo KR monochromatized
radiation (λ = 0.710 73 Å). Cell refinement and data reduction were
accomplished by the RAPID Auto program. The structure was then
solved with direct methods and refined using SHELXL-97 software
package.32 All non-hydrogen atoms were refined anisotropically. The
hydrogen atoms were located in the calculated positions and were
refined as constrained to bonding atoms. Relevant crystallographic
information is summarized in Table S4 in the Supporting Information,
and 50% thermal ellipsoid plots are shown in Figure 6. CCDC 791755
and 791756.
Cell Culture. NIH 3T3 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal calf
serum (FCS; Gibco) and 100 μg/mL penicillin/streptomycin
(Hyclone) at 37 °C in a 5:95 CO2/air incubator. The cells were cultured
3 days before dye loading onto 35-mm-diameter glass-bottomed cover-
slips. Then the cells were washed with PBS, bathed in PBS containing
10 μM probe DQCd1 for 30 min at 37 °C, washed with PBS three times
to remove the excess sensor, and bathed in PBS (2 mL) before imaging.
HEK293 cells were cultured in DMEM supplemented with 10% FCS
and 100 μg/mL penicillin/streptomycin at 37 °C in a 5:95 CO2/air
incubator. The cells were cultured 3 days before dye loading onto 35-
mm-diameter glass-bottomed coverslips. Then the cells were washed
with PBS, bathed in serum-free DMEM containing 10 μM probe
DQCd1 for 30 min at 37 °C, washed with PBS three times to remove
the excess sensor, and bathed in PBS (2 mL) before imaging.
Fluorescence Imaging. Confocal fluorescence imaging experi-
ments were performed with an Olympus FV-1000 laser-scanning
microscopy system, based on an IX81 inverted microscope (Olympus,
Japan). The microscope was equipped with multiple visible laser lines
(405, 458, 488, 515, 543, and 635 nm; continuous wave) and an
UPLSAPO 60ꢁ/NA 1.42 objective. Images were collected and pro-
cessed with Olympus FV10-ASW version 0107A-1 software.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: hjiang@iccas.ac.cn.
’ ACKNOWLEDGMENT
We thank the Chinese Academy of Sciences “Hundred
Talents Program”, the National Natural Science Foundation of
China (Grant 90813002), the National Basic Research Program
of China (No. 2011CB935800), and the Key Laboratory of
Pesticide & Chemical Biology, Ministry of Education, Central
China Normal University for financial support.
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