Phosphorescent Zinc Probe for Reversible Turn-On Detection with Bathochromically Shifted Emission

Article abstract of DOI:10.1021/acs.inorgchem.5b00967

Phosphorescent molecules are attractive complements to fluorescent compounds for bioimaging. Time-gated acquisition of the long-lived phosphorescence signals provides an effective means to eliminate unwanted background noises due to short-lived autofluorescence. We have previously investigated the molecular principles governing modulation of photoinduced electron transfer in phosphorescence zinc probes that were based on biscyclometalated Ir(III) complexes (Woo, H. et al. J. Am. Chem. Soc. 2013, 135, 4771-4787). The studies established that phosphorescence turn-on responses would be attainable for Ir(III) complexes with high triplet-state energies. This sets an upper limit to an emission wavelength, restricting the development of red- or near-IR-phosphorescence turn-on probes. To address this challenge, we designed and synthesized a new phosphorescent probe having an electron-deficient 2-(2-pyridyl)pyrazine diimine ligand tethering a di(2-picolyl)amine (DPA) zinc receptor. This ligand control led to red phosphorescence emission (λ_ems = 596 nm), with an excited-state reduction potential (E_red) retained as high as 1.44 V versus standard calomel electrode (SCE). The E_red value was more positive than the ground-state oxidation potential of DPA (1.05 V vs SCE), permitting an occurrence of photoinduced electron transfer at a rate of 2 × 10⁷ s^-1. Zinc binding at DPA abolished the electron transfer to produce phosphorescence turn-on signaling. The probe was capable of detecting zinc ions selectively over other competing biological metal ions in aqueous buffer solutions (pH 7.4, 20 mM piperazine-N,N′-bis(2-ethanesulfonic aid)) with the zinc dissociation constant of 109 pM. Finally, bioimaging utility of the probe has been successfully demonstrated by visualizing exogenously supplied zinc ions in live HeLa cells. The research described in this paper demonstrates that judicious ligand control enables retention of turn-on responses in the low-energy phosphorescence region.

Full text of DOI:10.1021/acs.inorgchem.5b00967

Article
pubs.acs.org/IC
Phosphorescent Zinc Probe for Reversible Turn-On Detection with
Bathochromically Shifted Emission
Seung Yeon Ryu,† Mijoung Huh,† Youngmin You,*,‡ and Wonwoo Nam*,†
‡
†Department of Chemistry and Nano Science and Division of Chemical Engineering and Materials Science, Ewha Womans
University, Seoul 120-750, Korea
S
* Supporting Information
ABSTRACT: Phosphorescent molecules are attractive com-
plements to fluorescent compounds for bioimaging. Time-
gated acquisition of the long-lived phosphorescence signals
provides an effective means to eliminate unwanted background
noises due to short-lived autofluorescence. We have previously
investigated the molecular principles governing modulation of
photoinduced electron transfer in phosphorescence zinc
probes that were based on biscyclometalated Ir(III) complexes
(Woo, H. et al. J. Am. Chem. Soc. 2013, 135, 4771−4787). The studies established that phosphorescence turn-on responses
would be attainable for Ir(III) complexes with high triplet-state energies. This sets an upper limit to an emission wavelength,
restricting the development of red- or near-IR-phosphorescence turn-on probes. To address this challenge, we designed and
synthesized a new phosphorescent probe having an electron-deficient 2-(2-pyridyl)pyrazine diimine ligand tethering a di(2-
picolyl)amine (DPA) zinc receptor. This ligand control led to red phosphorescence emission (λems = 596 nm), with an excited-
state reduction potential (E*red) retained as high as 1.44 V versus standard calomel electrode (SCE). The E*red value was more
positive than the ground-state oxidation potential of DPA (1.05 V vs SCE), permitting an occurrence of photoinduced electron
transfer at a rate of 2 × 107 s−1. Zinc binding at DPA abolished the electron transfer to produce phosphorescence turn-on
signaling. The probe was capable of detecting zinc ions selectively over other competing biological metal ions in aqueous buffer
solutions (pH 7.4, 20 mM piperazine-N,N′-bis(2-ethanesulfonic aid)) with the zinc dissociation constant of 109 pM. Finally,
bioimaging utility of the probe has been successfully demonstrated by visualizing exogenously supplied zinc ions in live HeLa
cells. The research described in this paper demonstrates that judicious ligand control enables retention of turn-on responses in
the low-energy phosphorescence region.
Marcus-normal region of electron transfer. On the basis of
these findings, it has been predicted that phosphorescence
emission of Ir(III) complexes with small ΔET cannot be
affected by PeT (Scheme 1a). Indeed, we observed the absence
of PeT for Ir(III) complexes exhibiting phosphorescence
emission at peak wavelengths longer than 517 nm (i.e.,
−ΔGPeT < 0 when ΔET < 2.4 eV).1 This lower bound of ΔET is
obviously greater than the case for PeT in fluorescence probes.
This thermodynamic loss is ascribed to exchange energy
(ΔEST) existing between singlet excited state (S1) and triplet
state (T1) (Scheme 1b).
There are two potential approaches to address the challenge:
One is to increase the E*red value of an Ir(III) complex, and the
other is to decrease the Eox value of a receptor. In the case of
the former approach, the modulation of E*red should rely on
control over Ered rather than ΔET to keep the emission in the
long-wavelength regions. We envisioned that such control
could be accomplished by incorporating an electron-deficient
diimine (N^N) ligand into biscyclometalated Ir(III) complexes.
I. INTRODUCTION
Phosphorescence modality offers unique advantages in
bioimaging, as the long-lived emission facilitates utilization of
time domains.1−3 Among the various classes of phosphorescent
molecules, cyclometalated Ir(III) complexes have been most
successful for bioimaging purposes.4−16 In particular, a range of
phosphorescent sensors that reversibly report the spatiotem-
poral fluctuations of biometals have been developed.3,17−21
Photoinduced electron transfer (PeT) is utilized to create the
metal ion probes, because the nonradiative PeT is several
orders faster than phosphorescence transition.22−29 Metal ion
binding abolishes the PeT, restoring phosphorescence
emission. We investigated the effectiveness of PeT for creation
of phosphorescence zinc probes, based on biscyclometalated
Ir(III) complexes and a di(2-picolyl)amino (DPA) zinc
receptor.1,2,30 The studies established that PeT from DPA to
the Ir(III) complexes adhered to the Rehm−Weller behavior
with −ΔGPeT = e·[Eox(DPA) − E*red(Ir)] + wp (−ΔGPeT
driving force for PeT; e, elementary charge; Eox, the ground-
state oxidation potential, wp, the ion-pairing term; E*red = Ered
,
+
ΔET, where Ered and ΔET are the ground-state reduction
potential and the triplet-state energy, respectively).24 Further
mechanistic studies revealed that the PeT was located in the
Received: April 29, 2015
© XXXX American Chemical Society
A
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a
vs standard calomel electrode (SCE)). Herein, we report the
design, synthesis, characterizations, and bioimaging applications
of a novel phosphorescence zinc probe (ZIrdap in Scheme 2).
The sensor produced zinc-induced phosphorescence turn-on
responses in the red-emission regions (λems = 596 nm) in
aqueous buffer solutions (pH 7.4, 20 mM piperazine-N,N′-
bis(2-ethanesulfonic acid) (PIPES)). Spectroscopic and electro-
chemical investigations revealed the occurrence of exoergic PeT
(−ΔGPeT = 0.39 eV), which was reversibly modulated by zinc
binding. Finally, bioimaging utility of the probe was successfully
demonstrated by phosphorescence visualization of exogenously
supplied zinc ions in live HeLa cells.
Scheme 1
II. RESULTS AND DISCUSSION
Synthesis of the Phosphorescence Zinc Sensor. The
synthetic route to the phosphorescence zinc sensor (ZIrdap) is
depicted in Scheme 2. The Pd(0)-catalyzed Stille reaction
between 5-amino-2-bromopyrazine and 2-(tributylstannyl)-
pyridine yielded the N^N ligand scaffold. Amidation of the
primary amine in the N^N ligand with α-bromoacetyl bromide,
followed by substitution of the α-bromide with di(2-picolyl)-
amine, yielded the DPA-appended N^N ligand (dap ligand,
hereafter). Finally, chelation of the dap ligand to the bis-
cyclometalated Ir(III) core produced the desired complex in an
overall 16% yield. Spectroscopic characterization data obtained
using multinuclear NMR spectroscopy and high-resolution
mass spectrometry were fully consistent with the proposed
structure. Reference complexes devoid of DPA and the amido
DPA moiety were also synthesized to investigate the origin of
the phosphorescence zinc responses (Irp and Irap; see Scheme
2 for the structures). Synthetic details and spectroscopic
identification data are summarized in the Experimental Section.
a
(a) The driving force for photoinduced electron transfer (−ΔGPeT
)
in blue- and red-phosphorescence sensors. (b) Comparison of −ΔGPeT
in a fluorescence sensor and a phosphorescence sensor. CS, singlet
charge-separated species; CS, triplet charge-separated species.
1
3
To validate this hypothesis, we chose to employ 2-(2-
pyridyl)pyrazine for the N^N ligand. The DPA zinc receptor
was introduced to the N^N ligand through an amide
linkage.31,32 We found that this ligand control indeed enabled
red phosphorescence emission with a large E*red value (1.44 V
Scheme 2. Synthesis of the Phosphorescent Zinc Sensor (ZIrdap) and Its Reference Compounds (Irp and Irap)
B
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Figure 1. (a) Phosphorescence spectra (dotted lines, CH3CN; solid lines, aqueous 20 mM PIPES solution at pH 7.4) of 10 μM zinc sensor (ZIrdap)
and its reference compounds (Irap and Irp) in the absence of zinc ion. Photoexcitation wavelength = 345 nm (ZIrdap and Irap) and 363 nm (Irp).
The asterisks (*) indicate peaks resulting from the second harmonic of the excitation beams. (b) The Lippert−Mataga plot of the phosphorescence
peak wavelengths of 10 μM ZIrdap, 10 μM Irap, and 10 μM Irp as a function of the solvent polarity parameter (f = (ε − 1)/(2ε + 1) − (n2 − 1)/
(2n2 + 1); ε and n are the dielectric constant and refractive index of a solvent, respectively).
Mechanism for Phosphorescence Responses of the
Zinc Sensor. UV−vis absorption spectra of ZIrdap and its
reference compounds carry the bands characteristic of bis-
cyclometalated Ir(III) complexes (Supporting Information,
Figure S1).1,33 ZIrdap (10 μM) exhibits orange phosphor-
escence in acetonitrile solutions with a peak emission
wavelength (λems) at 554 nm under photoexcitation at 345
nm (Figure 1a). Phosphorescence peak wavelengths of the two
reference compounds are found in the similar region (555 and
551 nm for Irap and Irp, respectively), while Irp possesses
pronounced vibronic structures. Of particular interest is the
bathochromic shifts of the phosphorescence spectra of ZIrdap
(λems = 596 nm) and Irap (λems = 591 nm) in aqueous buffer
solutions (20 mM PIPES, pH 7.4). Such a shift is not observed
for Irp. As shown in Figure 1b, Lippert−Mataga plots of the
phosphorescence peak wavelength energies as a function of the
solvent polarity parameter demonstrate the strong positive
solvatochromism for ZIrdap and Irap.34,35 These results suggest
Figure 2. Phosphorescence spectra (deaerated acetonitrile solutions)
of 10 μM ZIrdap in the absence (black) and presence (red) of 10
equiv of zinc ions and after the subsequent addition of 50 equiv of
TPEN (blue). The asterisk (*) indicates a peak resulting from a
harmonic of the excitation beam (345 nm).
that the amidation of the N^N ligand confers significant charge-
transfer character in the triplet state. Quantum chemical
calculations based on time-dependent density functional theory
(TD-DFT; CAM−B3LYP/LANL2DZ:6-311+G(d,p))//
B3LYP/LANL2DZ:6-311+G(d,p) predict an occurrence of
ligand-to-ligand charge-transfer transition, supporting this
analysis (Supporting Information, Figure S2).
The phosphorescence quantum yield (Φp) of ZIrdap (0.016)
is smaller than those of Irap (0.079) and Irp (0.10) in deaerated
CH3CN solutions, indicating that phosphorescence-quenching
processes are operative in ZIrdap. As shown in Figure 2,
addition of zinc ions (10 equiv in the form of Zn(ClO4)2) to
the solution containing 10 μM ZIrdap produces a ca. sixfold
enhancement in the phosphorescence intensity. Corresponding
Φp value for the zinc-bound ZIrdap approaches to 0.10. Of
interest is development of the distinct vibronic structures in the
phosphorescence emission of the zinc-bound form of ZIrdap.
This spectral signature is reminiscent of Irp (Figure 1a),
suggesting that zinc binding evokes structural changes in the
amide group. Actually, TD-DFT calculations show that N^N
ligand-centered π−π* transition becomes dominant in the
lowest triplet state upon zinc binding (Supporting Information,
Figure S2). Subsequent addition of the strong zinc chelator,
N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (TPEN), readily
restores the original spectrum, demonstrating excellent
reversibility. As expected, the reference complexes without
the DPA zinc receptors do not show zinc responses. Taken
together, the solution behaviors suggest that modulation of PeT
is most likely responsible for the phosphorescence turn-on
responses to zinc ions. Photophysical data for the Ir(III)
complexes are compiled in Table 1.
To estimate −ΔGPeT, electrochemical measurements using
cyclic and differential pulse voltammetry were performed
(Figure 3). The voltammograms of ZIrdap display two anodic
peaks at Eox = 1.05 and 1.60 V versus SCE, which correspond to
oxidation of the dap ligand and the Ir(III/IV) redox process,
respectively.1,2 The former and the latter waves are also found
in the dap ligand and Irap, respectively, confirming the
assignments. A reversible wave at E1/2 = −1.12 V versus SCE
due to the one-electron reduction of the N^N ligand is also
observed. The E*red value for the phosphorescent moiety in
ZIrdap can thus be calculated to be 1.44 V versus SCE, using
the ΔET value of 2.56 eV that was determined at the
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Table 1. Photophysical Data for the Phosphorescent Sensor and Its Reference Compounds
λabs (nm; ε × 10−4
λems
Eox
Ered
E*red
τ
kr
knr
kPeT
a
b
c
c
d
obse
f
g
h
M−1 cm−1
)
(nm)
Φp
(V vs SCE)
(V vs SCE) (V vs SCE)
(μs)
(× 104 s−1
)
(× 105 s−1
)
(× 105 s−1
)
a
j
ZIrdap
+ Zn2+
Irap
360 (1.98)
354 (2.26)
357 (1.65)
376 (1.66)
554,
0.016 1.05 (ir),
1.60 (qr)
−1.12 (r)
N.A.
1.44
N.A.
1.39
1.12
0.050
32
200
1.5
6.6
2.3
200
i
i
i
i
596
k
a
j
556,
572
0.10
0.079 1.63 (r)
0.10 1.49 (ir)
N.A.
6.5
1.5
6.6
0.25
N.A.
N.A.
N.A.
a
555,
591
−1.16 (r)
1.2
a
Irp
551,
552
−1.40 (qr)
40
a
b
10 μM phosphorescent compound in deaerated acetonitrile; 298 K. Phosphorescence quantum yield relatively determined by employing
c
fluorescein as the standard (0.1 N NaOH; Φf = 0.79). Determined by cyclic voltammetry. Conditions: scan rate = 100 mV/s; 1.00 mM in Ar-
saturated acetonitrile containing 100 mM Bu4NPF6 supporting electrolyte; a Pt wire counter and a Pt disc working electrodes; and a Ag/AgNO3
couple for the pseudo reference electrode. (r) = reversible wave, (qr) = quasi reversible wave, (ir) = irreversible wave, and (N.A.) = not applicable.
d
e
Excited-state reduction potential, E*red = Ered + ΔET. Phosphorescence lifetime for 50 μM compound in deaerated acetonitrile observed at λems
=
f
g
h
555 nm (λex = 377 nm). Radiative rate constant (kr) = Φp/τobs. Nonradiative rate constant (knr) = (1 − Φp)/τobs. Rate constant for PeT (kPeT),
kPeT = 1/τobs − 1/τobs(Zn), where τobs and τobs(Zn) are the observed phosphorescence lifetimes in the absence and presence of zinc ions, respectively.
i
N.A. indicates not applicable. 10 μM phosphorescent compound in air-equilibrated aqueous buffer solution (20 mM PIPES, pH = 7.4); 298 K.
j
Weighted-average phosphorescence lifetime of biexponential decays: zinc-free ZIrdap, 0.031 μs (53) and 0.79 μs (0.054); zinc-bound ZIrdap, 0.070
k
μs (0.58) and 7.2 μs (0.46). 5 equiv of zinc ions.
The above electrochemical data reveal thermodynamic
allowance (−ΔGPeT = 0.39 eV) for PeT in the zinc-free form
of ZIrdap. To monitor the PeT process, we acquired
phosphorescence decay traces at 555 nm of 50 μM ZIrdap
after nanosecond pulsed excitation at 377 nm (Figure 4). In the
Figure 3. Cyclic (solid lines) and differential pulse (dotted lines)
voltammograms of ZIrdap (blue), the dap ligand (gray), Irap (red),
and Irp (black). Conditions: scan rate = 100 mV s−1 (cyclic
voltammetry) and 4.0 mV s−1 (differential pulse voltammetry); 1.0
mM in Ar-saturated acetonitrile containing a 0.10 M Bu4NPF6
supporting electrolyte; a Pt wire counter electrode and a Pt disc
working electrode; a Ag/AgNO3 couple as the pseudo reference
electrode.
□
Figure 4. Photoluminescence decay traces of 50 μM Irp ( ), 50 μM
△
○
Irap ( ), and 50 μM ZIrdap ( ) in the absence (empty symbols) and
presence (filled symbols) of 5 equiv of zinc ions after nanosecond
pulsed photoexcitation at 377 nm. Conditions: deaerated CH3CN
solutions.
zinc-free state of ZIrdap, the transient phosphorescence signals
follow a biexponential decay model with time constants of
0.031 μs (53) and 0.79 μs (0.054) (values in the parentheses
are pre-exponential factors). Corresponding weighted-average
lifetime (τobs) is 0.050 μs. This lifetime value is significantly
shorter than those of 50 μM Irp (40 μs) and 50 μM Irap (1.2
μs). Addition of 5 equiv of zinc ions elongates the weighted-
average lifetime to 6.5 μs, implying that PeT is abolished by the
zinc binding. The PeT rate (kPeT) is estimated to be 2.0 × 107
s−1 from the relationship kPeT = 1/τobs − 1/τobs(zinc), where
τobs and τobs(zinc) are the phosphorescence lifetimes of ZIrdap
in the absence and presence of zinc ions, respectively. This kPeT
value is comparable with the rates for electron transfer
occurring between phenanthrene and dialkylamido groups in
the Z conformation.37 Note that the kPeT value is identical to
the overall nonradiative rate (knr, knr = (1 − Φp)/τobs = 2.0 ×
intersection between the UV−vis absorption and phosphor-
escence spectra. It is noted that ZIrdap retains the E*red value
similar to that (1.33−1.47 V vs SCE) of the previous blue-
phosphorescent zinc probes,1 despite the significant bath-
ochromic shift of 2450 cm−1 in the phosphorescence emission.
In addition, the E*red value is more positive than those of
biologically useful fluorophores, including fluorescein (1.15 V
vs SCE) and rhodamine B (1.25 V vs SCE),36 providing
improved margins for the occurrence of PeT from DPA.
Another notable feature is the high-lying oxidation potential of
the DPA moiety in ZIrdap (i.e., 1.05 V vs SCE).1 This value is
cathodically shifted from the oxidation potential of DPA in the
previous zinc sensor (1.28 V vs SCE), making PeT in ZIrdap
more exoergic.
D
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Figure 5. (a) Phosphorescence spectra of 10 μM ZIrdap in air-equilibrated water at different pH values. λex = 345 nm. (b) A plot of the integrated
(λems = 378−800 nm) phosphorescence intensity of 10 μM ZIrdap in air-equilibrated water as a function of pH.
107 s−1) within the experimental error. This finding supports
the notion that PeT is the dominant quenching process in the
zinc-free state of ZIrdap. In addition, the similarity between the
knr values of Irap (6.6 × 105 s−1) and the zinc-bound form of
ZIrdap (1.5 × 105 s−1) provides evidence for complete
suppression of PeT by zinc coordination.
Zinc Detection in the Aqueous Milieu. The phosphor-
escence intensity in the zinc-free form of 10 μM ZIrdap in air-
equilibrated aqueous buffer solutions at pH 7.4 (20 mM
PIPES) is higher than the case in acetonitrile. This increased
basal intensity can be ascribed to protonation of DPA. Indeed,
phosphorescence titration experiments demonstrate that the
integrated phosphorescence intensity starts to increase with
decreasing pH (Figure 5). The phosphorescence intensity
remains relatively large in the rage of pH 6−9, and decreases at
Figure 6. Phosphorescence turn-on responses of 10 μM ZIrdap to 5
equiv of zinc ions at various pH values.
pH < 6. The lower phosphorescence intensities at acidic pHs
may be ascribed to decoordination of the dap ligand, because
returning the pH to 7.4 does not restore the original spectrum.
Actually, the electrospray ionization mass spectrometry (ESI
corresponded to [Zn(ZIrdap−H+)(OH)(CH3CN)2]+ (calcd
MS; positive) spectrum of ZIrdap incubated at pH 4.0 for 1 h
displays peaks corresponding to the free dap ligand and the
biscyclometalated Ir complex fragment devoid of the dap ligand
(i.e., [Ir(C^N)2]+; Supporting Information, Figure S3). The
decoordination accompanies an appearance of a new
hypsochromically shifted emission band at 380−480 nm. The
spectral position and the photoluminescence lifetime of the
new emission coincide with those of the dap ligand (Supporting
Information, Figures S4 and S5), pointing to decoordination.
These data demonstrate that ZIrdap loses its ability to detect
zinc ions under prolonged exposure to acidic environments.
Decoordination dynamics appears to be slow, but further
investigations would be necessary to establish the kinetics of
decoordination. Still, addition of 5 equiv of ZnCl2 produces
twofold enhancements in the phosphorescence intensities in
the range of pH 5.0−8.0 (Figure 6).
The phosphorescence zinc responses could be distinguish-
able from the proton-induced changes, because a hypsochromic
shift of 700 cm−1 is evoked by zinc binding (Supporting
Information, Figure S6; zinc-free state, λem = 596 nm; zinc-
bound state, λem = 572 nm). The zinc-induced hypsochromic
shift in the phosphorescence spectrum may originate from
amido-to-iminol tautomerization and binding of HO− ion at the
zinc center, as recently reported by Shiraishi and co-workers.32
Actually, ESI MS (positive mode) spectra of the zinc-bound
form of ZIrdap contained a peak at m/z = 1146.0 that
1146.2), supporting this hypothesis (Supporting Information,
Figure S7). Furthermore, TD-DFT results for this structure
predicted a hypsochromic shift (645 cm−1) similar to the
experimental observation (Supporting Information, Figures
S2). Such a hypsochromic shift upon zinc binding is not
observed in acetonitrile solutions.
Phosphorescence zinc titration for 10 μM ZIrdap was
performed with increasing the total zinc concentration from 0
to 15 μM at a 0.05 μM interval (Figure 7a). The
phosphorescence intensity increases in proportion with the
added zinc concentration and levels off after 1.0 equiv of ZnCl2.
Job’s plot analysis supports this 1:1 binding stoichiometry
(Supporting Information, Figure S8). Corresponding zinc
titration isotherm was fit to the analytical equations of the
phosphorescence intensity and the free zinc concentration (eqs
1 and 2 in the Experimental Section). Iterative nonlinear least-
squares fit to the equations returns the zinc dissociation
constant (Kd) of 109 pM (Figure 7b). This value is 2 orders of
magnitude smaller than the Kd value of the previous
phosphorescence zinc sensor, indicating tight zinc binding of
ZIrdap.2 The stronger binding capability may originate from
participation of the amide group in zinc coordination.31,32
As shown in Figure 8, the phosphorescence response of
ZIrdap is selective to zinc ions over other biological metal ions.
Presence of 100 mM Na, 100 mM Mg, 100 mM K, 100 mM
Ca, 50 μM Cr, 50 μM Mn, and 50 μM Fe(III) ions has no
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Figure 8. Phosphorescence zinc selectivity of 10 μM ZIrdap over other
competing metal ions at pH 7.4 (20 mM PIPES containing 100 mM
KCl): 100 mM Na, Mg, and Ca, and 50 μM Cr, Mn, Fe(II), Fe(III),
Co, Ni, Cu, and Cd. 50 μM ZnCl2 was added to the ZIrdap solution
containing the competing metal ions.
Figure 7. (a) Phosphorescence zinc response of 10 μM ZIrdap at pH
7.4 (air-equilibrated aqueous solution buffered with 20 mM PIPES).
The asterisk (*) indicates a peak resulting from the second harmonic
of the excitation beam (345 nm). (inset) Titration isotherm of the
integrated phosphorescence intensity as a function of the total zinc
concentration. (b) Titration isotherm of the integrated phosphor-
escence intensity as a function of the calculated concentration of free
zinc. Red curve is the nonlinear least-squares fit of the titration data to
eq 1 in the Experimental Section.
Figure 9. Phosphorescence spectra of 10 μM ZIrdap (20 mM PIPES,
pH 7.4) in the absence (black) and presence (blue) of 1.5 equiv of
CdCl2 and 1.5 equiv of ZnCl2 (red). λex = 345 nm. The asterisk is due
to the second harmonic of the excitation beam.
motif. Indeed, UV−vis absorption titration of ZIrdap (20 mM
PIPES, pH 7.4) with increasing either ZnCl2 or CdCl2
concentrations revealed different spectral profiles. As shown
in Figure 10, the metal-free ZIrdap exhibits the lowest-energy
absorption band at 346 nm. The additions of CdCl2 and ZdCl2
lead to bathochromic shifts of the absorption band with
multiple isosbestic points (CdCl2, 358, 318, and 303 nm;
ZnCl2, 365, 301, and 273 nm). These red shifts are opposite to
the blue shifts in the phosphorescence spectra (Figure 9),
suggesting that polar ground states are formed upon metal
binding. In the case of the cadmium titration, the absorbance at
337 nm decreases in a monotonic manner and reached a
plateau after 1 equiv of Cd ions (Figure 10a,c). Similar linear
behaviors are observed in the phosphorescence cadmium
titration experiments (Figure 11). In sharp contrast, zinc
titration produces two-stage spectral changes (Figure 10b,c).
The incremental addition of ZnCl2 up to 0.5 equiv yields
absorption changes identical to the spectral signatures of the
cadmium titration. Further addition of ZnCl2 evokes the second
absorption responses with a hypsochromic shift in the peak
wavelength. An isosbestic point at 349 nm is observed,
indicating that the second response is not due to instrumental
influence on the phosphorescence zinc (50 μM) response of 10
μM ZIrdap. Weaker responses are observed when 50 μM
Fe(II) ions are present, and paramagnetic ions, including Co,
Ni, and Cu, abolish the ability of ZIrdap for phosphorescence
zinc detection. Similar to other zinc probes,38−54 Cd binding
produces turn-on responses. However, the interferences by
these metal ions will be minimal due to low concentrations of
the chelatable forms in the biological milieu.
Comparison of Zinc and Cadmium Binding Behaviors.
Many zinc probes experience interferences by cadmium ions.
ZIrdap also displays a phosphorescence turn-on response to Cd
ions. However, as shown in Figure 9, the phosphorescence
spectrum of the cadmium-bound form differs from those of
zinc-free and -bound forms. The peak wavelength of the
phosphorescence emission of the cadmium-bound form is 561
nm, which shifts hypsochromically from the zinc-free state by
1200 cm−1. The extent of the blue shift is greater than the case
for zinc binding (700 cm−1). Another notable feature in the
phosphorescence spectrum for the cadmium-bound form is the
presence of a shoulder peak at 526 nm. This spectral signature
is indicative of a Cd-binding mode different from a Zn-binding
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Figure 10. UV−vis absorption titration of 10 μM ZIrdap with CdCl2 (a) and ZnCl2 (b) in 20 mM PIPES buffer at pH 7.4, and corresponding
titration isotherms for cadmium and zinc monitored at 337 nm (c).
of zinc and cadmium ions (Supporting Information, Figure 9).
Although the complexity of the NMR spectra hampers
complete assignments, the NMR spectra may suggest that the
Cd-bound form retains the amido tautomer of the metal-free
ZIrdap, whereas zinc binding induces the amido-to-iminolate
tautomerization. Several attempts to obtain crystals for the
metal-bound forms were not successful. On the basis of the
results, it is proposed that ZIrdap adopts a predominant 2:1
(ZIrdap/Zn) binding geometry at low zinc concentrations (i.e.,
<0.5 equiv), where the amido form of ZIrdap is involved. At
high zinc concentrations, a 1:1 complex having the iminolate
form becomes the dominant species. This hypothesis is
supported by the ESI MS and TD-DFT results for the zinc-
bound ZIrdap (Supporting Information, Figures S2 and S7).
However, further investigations should be performed to fully
elucidate the structure−photophysical response relationship,
because the phosphorescence zinc titration exhibits the one-
stage (i.e., monotonic) increase in the phosphorescence
intensity (Figure 7).
Finally, the cadmium dissociation constant (Kd) was
determined using the phosphorescence titration results. Non-
linear least-squares fit of the titration data to eqs 1 and 2 in the
Experimental Section yields a Kd value of 82 pM. This value is
comparable to the Kd for zinc binding, implying that ZIrdap
binds cadmium ions as strong as zinc ions.
Visualization of Intracellular Zinc. Having established
the ability of ZIrdap for phosphorescence zinc detection, we
sought to demonstrate bioimaging utility of the zinc probe
Figure 11. (a) Phosphorescence cadmium response of 10 μM ZIrdap
at pH 7.4 (air-equilibrated aqueous solution buffered with 20 mM
PIPES). The asterisk (*) indicates a peak resulting from the second
harmonic of the excitation beam (345 nm). (inset) Titration isotherm
of the integrated phosphorescence intensity as a function of the total
cadmium concentration. (b) Titration isotherm of the integrated
phosphorescence intensity as a function of the calculated concen-
tration of free cadmium. Red curve is the nonlinear least-squares fit of
the titration data to eq 1 in the Experimental Section.
(Figure 12). HeLa, human cervical cancer cells, were employed
for the intracellular zinc imaging experiments. ZIrdap is
permeable through the membrane of HeLa cells, as evidenced
by phosphorescence halos (λex = 405 nm and λems = 441−691
nm) in the cytosol of HeLa cells pretreated with 10 μM ZIrdap
(10 min). The spectral profile of the intracellular phosphor-
escence signals resembles the phosphorescence spectrum of the
zinc-bound form of ZIrdap recorded in acetonitrile (Figure
12c), confirming that the ZIrdap is responsible for the
photoemission.55 The non-negligible phosphorescence inten-
sity might be due to protonation or endogenous zinc levels in
HeLa cells comparable to the Kd value of ZIrdap.56 Addition of
the ionomeric form of zinc ions (50 μM ZnCl2 + 100 μM
sodium pyrithione) leads to a prompt increase in the
phosphorescence intensity (Figure 12a). The corresponding
drifts. The titration isotherm plotting the absorbance at 337 nm
versus the added ZnCl2 concentration reveals the zinc response
that is different from the cadmium case (Figure 10c).
1
To obtain insight into the binding geometries, H NMR
spectra were obtained for ZIrdap in the absence and presence
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Figure 12. Phosphorescence visualization of exogenously supplied zinc ions in the form of ZnPT (ZnCl2/sodium pyrithione = 1:2, mol/mol). (a)
From the left: bright field image, HeLa cells incubated with 10 μM ZIrdap for 10 min, cells subsequently treated with 50 μM ZnPT, after the
successive treatment with 100 μM TPEN. λem = 441−691 nm (λex = 405 nm). Scale bar = 100 μm. (b) Average photoluminescence intensities of the
HeLa cells treated with 10 μM ZIrdap (left) and after subsequent treatment with 50 μM ZnPT (middle), followed by 100 μM TPEN addition
(right). The photoluminescence intensities of the ZnPT-treated cell images were normalized to 1. Averages of 11 experiments. (c) Comparison of
the phosphorescence spectrum (gray line) of the zinc-bound ZIrdap (CH3CN) and the λ-profiles of the photoluminescence emission from the
●
△
ZIrdap-treated HeLa cells before ( ) and after ( ) the ZnPT addition. More photoluminescence cell images are included in the Supporting
Information, Figure S10.
phosphorescence turn-on ratio as quantitated by an average of
11 dishes is 1.5 (Figure 12b), barely enabling zinc imaging.
Validity of the phosphorescence turn-on responses was assessed
by the fluorescence-activated cell sorting (FACS) analyses
(Supporting Information, Figure S11). Subsequent addition of
100 μM TPEN reduces the phosphorescence intensities,
confirming zinc responses.
and 2-(2-pyridyl)pyrazine. This ligand structure had two effects
on the thermodynamic parameters of PeT: One was increasing
E*red of the phosphorescent center, and the other was
decreasing Eox of DPA. The net result of these two effects
was exothermicity of PeT (−ΔGPeT = 0.39 eV) at a significantly
small phosphorescence energy (λems = 596 nm). We monitored
the occurrence of the intramolecular PeT and determined the
PeT rate to be 2.0 × 107 s−1, by employing transient
photoluminescence techniques. This PeT rate was virtually
identical to the overall rate of nonradiative transition in the
zinc-free form of ZIrdap, indicating the effectiveness of PeT in
the phosphorescence modulation. The zinc probe produced
turn-on responses, which were fully reversible and selective to
zinc ions over other biological metal ions. However, similar
turn-on phosphorescence signaling was evoked by cadmium
ion, while titration experiment results suggested different metal-
binding modes. Finally, the probe was capable of visualizing
exogenously supplied zinc ions in live HeLa cells. Despite the
advance, however, one challenge to attenuation of 1O2-
mediated phototoxicity remains. This calls for future studies
One drawback of using ZIrdap in live cell imaging is noted:
ZIrdap is cytotoxic to HeLa cells upon prolonged exposure
(>30 min) to the photoexcitation beam. We speculated that
this phototoxicity might result from photosensitization of
singlet dioxygen (1O2) by ZIrdap.57 Indeed, the quantum yields
for singlet oxygen photogeneration (ΦΔ) of both zinc-free and
zinc-bound forms of ZIrdap, determined using methylene blue
(ΦΔ = 0.52)58 and 1,3-diphenylisobenzofuran59 as the standard
1
and the O2-selective substrate, respectively, approach unity
(Supporting Information, Figure S12). The ΦΔ values for Irap
and Irp are also as high as 0.89 and 0.68, respectively, indicating
that the cyclometalated Ir(III) complexes are responsible for
1
the O2 photosensitization. These results present an additional
1
challenge to the establishment of molecular strategies to
attenuate phototoxicity of phosphorescent probes.
to establish molecular strategies for the minimization of O2
sensitization.
III. SUMMARY AND CONCLUSIONS
IV. EXPERIMENTAL SECTION
It has remained as a significant challenge to develop red-
phosphorescent probes that can reversibly detect biological zinc
ions. This difficulty stems from the thermodynamic forbiddance
of PeT in the probes with low-energy phosphorescence
emission. We have developed a zinc probe (ZIrdap) that is
capable of producing fully reversible turn-on responses in the
low-energy phosphorescence regions. The molecular construct
for the phosphorescent probe involved the biscyclometalated
Ir(III) complex and the DPA-appended 2-(2-pyridyl)pyrazine
N^N ligand. An amide linkage was employed for bridging DPA
Materials and Synthesis. Caution! Perchlorate salts of metal
complexes are potentially explosive. Only small quantities of material
should be handled with care. The chloride-bridged Ir(III) precursor
[(dfppy)2Ir(μ-Cl)]2 (dfppy = 2-(2,4-difluorophenyl)pyridinate) was
synthesized according to a literature method.60 Commercially available
chemicals were used as received. All glassware and magnetic stirring
bars were thoroughly dried in a convection oven. Reactions were
monitored using thin layer chromatography (TLC). Commercial TLC
plates (silica gel 254, Merck Co.) were developed, and the spots were
visualized under UV light at 254 or 365 nm. Silica gel column
chromatography was performed with silica gel 60 G (particle size
H
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0.063−0.200 mm, Merck Co.). H, 13C, and 19F NMR spectra were
collected with Bruker Ultrashield 300 and 400 Plus NMR
spectrometers and referenced to residual proton peaks of the
deuterated solvent. Mass spectra were recorded using a Thermo
Electrons Co. Finnigan LCQ Advantage Max spectrometer and a
JEOL JMS-600W mass spectrometer. Elemental analysis was
performed using CE Instrument EA1110 and EA1112 for C, H, and N.
2-Amino-5-(2-pyridyl)pyrazine. 2-Amino-5-bromopyrazine
(2.60 g, 17.0 mmol), 2-(tributylstannyl)pyridine (6.19 g, 19.0
mmol), and tetrakis(triphenylphosphine)palladium(0) (1.76 g, 1.70
mmol) were dissolved in toluene (120 mL), and the solution was
refluxed for 1 d under an Ar atmosphere. After it cooled to room
temperature, the reaction mixture was concentrated and subjected to
chromatographic purification on silica gel column (CH2Cl2/CH3OH =
1H). 13C NMR (CDCl3, 100 MHz) δ: 59.56, 60.50, 114.04, 122.73,
122.80, 123.83, 124.01, 124.77, 127.70, 134.98, 136.96, 139.32, 140.43,
144.50, 145.85, 148.84, 149.03, 149.51, 149.82, 151.99, 152.76, 154.22,
157.72, 172.69, 206.97. 19F NMR (CDCl3, 376 MHz) δ: −108.25 (m,
2F), −105.02 (m, 2F), −73.00 (d, J = 771 Hz, 6F). HR MS (FAB,
positive, m-NBA): Calcd for C45H33F4IrN9O ([M−PF6]+), 984.2373;
found: 984.2370.
1
Irp. The identical method for the synthesis of ZIrdap was
employed, except using 2-amino-5-(2-pyridyl)pyrazine instead of 2-
((di(2-picolyl)amino)acetylamino)-5-(2-pyridyl)pyrazine. 0.210 g
1
(29%). H NMR (CD2Cl2, 400 MHz) δ: 5.65 (s, 2H), 5.74 (t, J =
9.7 Hz, 2H), 6.64 (t, J = 9.2 Hz, 2H), 7.09 (t, J = 7.2 Hz, 1H), 7.15 (t,
J = 7.3 Hz, 1H), 7.29 (s, 1H), 7.39 (t, J = 6.8 Hz, 1H), 7.48 (d, J = 5.8
Hz, 1H), 7.70 (d, J = 5.7 Hz, 1H), 7.85−7.89 (m, 3H), 8.08 (t, J = 8.0
Hz, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.31−8.37 (m, 2H), 9.10 (s, 1H).
13C NMR (CD2Cl2, 100 MHz) δ: 99.38, 99.65, 99.94, 100.21, 114.18,
114.36, 114.49, 114.66, 122.21, 124.13, 124.21, 124.30, 124.42, 124.63,
126.93, 127.93, 128.12, 132.15, 138.92, 139.78, 139.87, 140.10, 145.90,
148.99, 149.20, 150.52, 152.72, 153.51, 155.28, 158.20, 160.72, 164.31,
164.83, 165.47. 19F NMR (CD2Cl2, 376 MHz) δ: −108.69 (m, 2F),
−106.13 (m, 2F), −73.03 (d, J = 768 Hz, 6F). HR MS (FAB, positive,
m-NBA): Calcd for C31H20F4IrN6 ([M−PF6]+), 745.1315; found:
745.1312.
1
49:1 to 9:1, v/v). Yellowish-brown powder (2.30 g, 78%). H NMR
(CDCl3, 400 MHz) δ: 4.71 (s, 2H), 7.23−7.25 (m, 1H), 7.79 (t, J =
7.7 Hz, 1H), 8.03 (d, J = 1.4 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.64
(d, J = 4.8 Hz, 1H), 9.06 (d, J = 1.4 Hz, 1H). 13C NMR (CDCl3, 100
MHz) δ: 119.73, 122.79, 130.83, 136.83, 140.81, 141.71, 149.17,
154.16, 154.98. HR MS (EI, positive): Calcd for C9H8N4, 172.0749;
found: 172.0746. Anal. Calcd for C9H8N4: C, 62.78; H, 4.68; N, 32.54.
Found: C, 62.71; H, 4.72; N, 32.21%.
2-(Bromoacetylamino)-5-(2-pyridyl)pyrazine. 2-Amino-5-(2-
pyridyl)pyrazine (0.500 g, 2.90 mmol) and K2CO3 (0.480 g, 3.50
mmol) were dissolved in an anhydrous acetonitrile (50 mL).
Bromoacetyl bromide (276 μL, 3.20 mmol) was added to a stirred
solution of the reaction mixture, which was stirred additionally for 16 h
at room temperature. After removal of the solvent, the concentrated
mixture was subjected to silica gel column chromatography (CH2Cl2
to CH2Cl2/CH3OH = 9:1, v/v) to give a yellow powder (0.460 g,
2-(Acetylamino)-5-(2-pyridyl)pyrazine. An anhydrous acetoni-
trile solution (30 mL) containing 2-amino-5-(2-pyridyl)pyrazine
(0.150 g, 0.870 mmol) and K2CO3 (0.220 g, 0.159 mmol) was stirred
at room temperature for 30 min. Acetyl bromide (97.0 μL, 1.30 mmol)
was added to the solution, which was stirred for additional 4 h. The
reaction mixture was concentrated under reduced pressure and
subjected to column purification on silica gel (CH2Cl2 to CH2Cl2/
1
1
54%). H NMR (CDCl3, 400 MHz) δ: 4.08 (s, 2H), 7.33−7.36 (m,
CH3OH = 19:1, v/v). Yellowish-white powder (0.0690 g, 37%). H
1H), 7.86 (t, J = 7.6 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.70 (d, J = 4.8
Hz, 1H), 9.36 (d, J = 1.2 Hz, 1H), 9.53 (d, J = 1.2 Hz, 1H). 13C NMR
(deuterated dimethyl sulfoxide (DMSO), 100 MHz) δ: 31.12, 61.94,
122.22, 125.80, 135.43, 141.58, 148.05, 149.58, 150.95, 165.46, 172.57.
HR MS (EI, positive): Calcd for C11H9N4OBr, 291.9960; found:
291.9957. Anal. Calcd for C11H9N4OBr: C, 45.07; H, 3.09; N, 19.11.
Found: C, 44.82; H, 3.06; N, 19.29%.
NMR (CDCl3, 400 MHz) δ: 2.28 (s, 3H), 7.31−7.34 (m, 1H), 7.85 (t,
J = 7.6 Hz, 1H), 7.91 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.69 (d, J = 4.8
Hz, 1H), 9.29 (d, J = 1.2 Hz, 1H), 9.55 (s, 1H). 13C NMR (CDCl3,
100 MHz) δ: 24.50, 120.99, 121.86, 123.92, 135.12, 137.11, 140.45,
146.98, 147.51, 149.28, 149.63, 168.40. HR MS (CI, positive): Calcd
for C11H11N4O ([M + H]+), 215.0933; found: 215.0938. Anal. Calcd
for C11H10N4O: C, 61.67; H, 4.71; N, 26.15. Found: C, 61.43; H, 4.68;
N, 25.97%.
2-((Di(2-picolyl)amino)acetylamino)-5-(2-pyridyl)pyrazine.
An acetonitrile solution (50 mL) containing 2-(bromoacetylamino)-5-
(2-pyridyl)pyrazine (0.194 g, 0.662 mmol), di(2-picolyl)amine (0.120
g, 0.602 mmol), and K2CO3 (0.128 g, 0.927 mmol) was stirred for 1 d
at room temperature. The reaction mixture was filtered to remove
residual K2CO3, and the filtrate was concentrated. Chromatographic
purification on silica gel column (CH2Cl2 to CH2Cl2/CH3OH = 9:1,
Irap. The identical method for the synthesis of ZIrdap was
employed, except using 2-(acetylamino)-5-(2-pyridyl)pyrazine instead
of 2-((di(2-picolyl)amino)acetylamino)-5-(2-pyridyl)pyrazine. 0.560 g
1
(37%). H NMR (CD2Cl2, 400 MHz) δ: 2.11 (s, 3H), 5.64−5.70 (m,
2H), 6.52−6.62 (m, 2H), 6.97−7.04 (m, 2H), 7.39 (d, J = 5.8 Hz,
1H), 7.42−7.46 (m, 1H), 7.49 (d, J = 5.8 Hz, 1H), 7.80 (t, J = 7.2 Hz,
2H), 7.92 (d, J = 5.2 Hz, 1H), 8.11 (t, J = 7.6 Hz, 1H), 8.27 (d, J = 8.4
Hz, 2H), 8.36 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.84 (d, J = 1.2 Hz,
1H), 9.33 (d, J = 1.2 Hz, 1H). 13C NMR (CD2Cl2, 100 MHz) δ: 24.74,
99.93, 114.61, 124.42, 124.54, 128.76, 135.87, 140.02, 140.11, 140.72,
145.35, 145.49, 149.25, 149.42, 151.18, 152.55, 154.32, 169.67. 19F
NMR (CD2Cl2, 376 MHz) δ: −108.74 (m, 2F), −106.08 (m, 2F),
−73.02 (d, J = 770 Hz, 6F). HR MS (FAB, positive, m-NBA): Calcd
for C33H22F4IrN6O ([M−PF6]+), 787.1420; found: 787.1421. Anal.
Calcd for C33H22F10IrN6OP: C, 42.54; H, 2.38; N, 9.02. Found: C,
42.82; H, 2.45; N, 9.04%.
1
v/v) gave a brown powder (0.220 g, 88%). H NMR (CDCl3, 400
MHz) δ: 3.59 (s, 2H), 4.04 (s, 4H), 7.17−7.20 (m, 2H), 7.30−7.32
(m, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.66 (t, J = 7.6 Hz, 2H), 7.84 (t, J =
7.6 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 4.8 Hz, 2H), 8.70
(d, J = 4.8 Hz, 1H), 9.40 (d, J = 1.6 Hz, 1H), 9.54 (d, J = 1.2 Hz, 1H).
13C NMR (CDCl3, 100 MHz) δ: 59.06, 60.80, 120.92, 122.56, 123.15,
123.71, 135.32, 136.77, 137.05, 140.88, 146.42, 148.34, 149.29, 149.48,
154.47, 157.94, 171.18, 207.01. HR MS (FAB, positive, m-NBA):
Calcd for C23H21ON7, 411.1808; found: 411.1818.
ZIrdap. An anhydrous CH2Cl2 (40 mL) solution containing the
chloride-bridged Ir(III) dimer ([(dfppy)2Ir(μ-Cl)]2; 0.350 g, 0.290
mmol) and 2-((di(2-picolyl)amino)acetylamino)-5-(2-pyridyl)-
pyrazine (0.200 g, 0.490 mmol) was refluxed for 6 h under an Ar
atmosphere. The reaction mixture was cooled to room temperature,
and NH4PF6 (15 equiv) was slowly added to the solution. After 12 h,
the reaction mixture was filtered to remove NH4PF6 and concentrated
under vacuum. The crude mixture was subjected to flash column
chromatography on silica gel with CH2Cl2 to CH2Cl2/CH3OH = 19:1
(v/v). Further purification by preparative TLC techniques was
performed to isolate an orange powder (0.290 g, 44%). 1H NMR
(CD3CN, 300 MHz) δ: 3.50 (d, J = 3.6 Hz, 2H), 3.97 (s, 4H), 5.71
(m, 2H), 6.68−6.76 (m, 2H), 7.07−7.20 (m, 4H), 7.26 (d, J = 7.8 Hz,
2H), 7.51 (m, 1H), 7.62 (m, 3H), 7.73 (m, 1H), 7.90−7.99 (m, 3H),
8.17 (td, J = 7.8, 1.5 Hz, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.52 (d, J = 8.1
Hz, 1H), 8.57 (m, 2H), 8.85 (d, J = 1.2 Hz, 1H), 9.50 (d, J = 1.2 Hz,
Spectroscopic Measurements. Milli-Q grade water (18.2 MΩ·
cm) was used to prepare solutions for spectroscopic measurements.
PIPES (≥ 99%) was purchased from Aldrich. A pH 7.4 buffer solution
was prepared by dissolving PIPES (20 mM) in milli-Q water and
adjusting the pH with standard KOH solution (45 wt %, Aldrich) or
concentrated HCl (Aldrich). The buffer solution was further treated
with Chelex 100 resin (BIO-RAD) to remove trace metal ions, filtered
through a membrane (pore size = 0.45 μm), and its pH was
reexamined prior to use. Fresh metal stock solutions (typically, 0.10 or
0.010 M except for CrCl3·6H2O) were prepared in milli-Q water using
the corresponding chloride salts: CuCl2 (99.999%, Aldrich), NaCl
(≥99.5%, Aldrich), KCl (puratonic grade, Calbiochem), MgCl2
(99.99%, Aldrich), CaCl2 (99.99%, Aldrich), CrCl3·6H2O (98%,
Aldrich), MnCl2 (99.99%, Aldrich), FeCl2 (99.99%, Aldrich), CoCl2
(99.9%, Aldrich), NiCl2 (99.99%, Aldrich), CdCl2 (99.99%, Aldrich),
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and ZnCl2 (99.999%, Aldrich). A TPEN solution was prepared by
dissolving N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (≥99%,
Sigma) in DMSO (99.9%, Aldrich). Zn(ClO4)2·6H2O (Aldrich) was
dissolved in CH3CN (spectrophotometric grade, Aldrich) to 1.0, 10,
and 100 mM concentrations. The Ir(III) complex solutions were
prepared by dissolution in CH3CN to concentrations of 10 mM, 1.0
mM, 100 μM, and 10 μM. The 10 μM solutions were used for
spectroscopic measurements, otherwise stated. For aqueous solutions,
3.0 mL of the PIPES buffer and 3 μL of the Ir(III) complex solution
(10 mM in DMSO) were mixed to give a 10 μM solution. A 1 cm × 1
cm fluorimeter cell (Hellma) with a rubber cap was used for the
steady-state optical measurements. UV−vis absorption spectra were
collected on a Varian Cary 50 spectrophotometer at room temper-
ature. Phosphorescence spectra were obtained using a Quanta Master
40 scanning spectrofluorimeter at room temperature. The photo-
luminescence quantum yields (Φ) were relatively determined
according to following standard equation: Φ = Φref × (I/Iref) ×
(Aref/A) × (n/nref)2, where A, I, and n are the absorbance at the
excitation wavelength, integrated photoluminescence intensity, and the
refractive index of the solvent, respectively. Fluorescein as an aqueous
0.1 N NaOH solution was used as the external reference (Φref = 0.79).
The refractive index of the 0.1 N NaOH solution was assumed to be
identical to the value for pure water. The 10 μM solutions were
deaerated by bubbling Ar prior to performing the measurements. Ar-
saturated 50 μM solutions (CH3CN) were used for the determination
of the phosphorescence lifetimes (τobs). Photoluminescence decay
traces were acquired based on time-correlated single photon counting
techniques using a FluoTime 200 instrument (PicoQuant, Germany).
A 377 nm diode laser (PicoQuant, Germany; pulse energy = 35 pJ)
with a repetition rate of 125 kHz was used as the excitation source.
The signals at 555 nm were obtained using an automated motorized
monochromator, and recorded with a NanoHarp unit. The decay
profiles were analyzed (OriginPro 8.0, OriginLab) using a single or
double exponential decay model.
fit could not be improved. A nonlinear curve fitting module embedded
in OriginPro 8.5 (OriginLab) was used for this purpose.
Cell Culture. HeLa cells were cultured in DMEM supplemented
with 10% fetal bovine serum and penicillin (100 units/mL) at 37 °C in
a humidified incubator under 5% CO2. One day before imaging, cells
were passed and plated onto poly(D-lysine)-coated glass-bottom
culture dishes.
Confocal Laser Scanning Microscopy. After the cells were
washed with fresh DMEM, a 10 μM ZIrdap in DMSO (biotech grade,
Aldrich) was added to the culture media. The cells were incubated for
10 min at 37 °C. The incubated cells were washed twice with fresh
DMEM (serum-free), and photoluminescence micrographs were taken
using a Carl Zeiss LSM 510 META confocal laser scanning microscope
using a Newport MaiTai eHP DeepSee multiphoton excitation system.
An excitation beam (405 nm) was focused onto the dish, and the
signals were acquired through 30 emission channels covering the range
of 441−691 nm. The cells were imaged after subsequent treatment
with 50 μM ZnCl2/NaPT. Finally, 100 μM TPEN (DMSO) was
introduced. ZnPT and TPEN were added directly into the culture
media. Exposure to the excitation beam was kept as short as possible
(<10 min) to minimize photoinduced cell death. Photoluminescence
images and mean intensities were processed using the ZEN and
ImageJ software, respectively.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b00967.
1H, 13C, and 19F NMR spectra, UV−vis absorption
spectra, a summary of TD-DFT calculation results, pH
titration results, FACS analysis data, phosphorescence
zinc responses at different pH values, ESI MS spectrum
of the zinc-bound from of ZIrdap, 1H NMR titration with
varying the zinc concentration, additional 1H NMR
spectra of ZIrdap in different solvents, phosphorescence
Job’s plot, phosphorescence responses to cadmium ions,
more cell images, Cartesian coordinates of optimized
geometries, and determination of the quantum yields for
1O2 photosensitization. (PDF)
Electrochemical Measurements. Cyclic and differential pulse
voltammetry experiments were performed using a CHI630 B
instrument (CH Instruments, Inc.) using three-electrode cell
assemblies. A Pt wire and a Pt disc were used as the counter and
working electrodes, respectively. A Ag/AgNO3 couple was used as a
pseudo reference electrode. Measurements were performed in Ar-
saturated CH3CN (3 mL) using tetra-n-butylammonium hexafluor-
ophosphate (Bu4NPF6) as the supporting electrolyte (0.10 M) at scan
rates of 100 mV s−1 (cyclic voltammetry) and 4 mV s−1 (differential
pulse voltammetry). The concentration of the Ir(III) complex was 1.0
mM. A ferrocenium/ferrocene reference was employed as the external
reference.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: odds2@ewha.ac.kr. (Y.Y.)
*E-mail: wwnam@ewha.ac.kr. (W.N.)
■
Determination of Kd. An approach previously reported by us2,61
was used to determine Kd values for Zn(II) and Cd(II) binding to
ZIrdap. An equilibrium model for formation of a 1:1 ligand/metal
complex was applied. Mass balance equations for the total
concentrations of sensor and metal ions (M2+; M = Zn or Cd) can
be expressed by eqs 1 and 2.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
phosphorescence intensity(P. I. )
= [ZIrdap]total × (α1Kd + α2[M2+
Authors acknowledge the financial support from CRI (NRF-
2012R1A3A2048842 to W.N.), GRL (NRF-2010-00353 to
W.N.), GFP (CISS-2012M3A6A6054204 to Y.Y.), and the
Ewha Womans Univ. (1-2015-0447-001-1 to Y.Y.).
]
free )/(Kd + [M2+
]
)
free
(1)
(2)
2
[M2+
]
+ ([ZIrdap]total + Kd − [M2+ total )[M2+
] ]
free
free
REFERENCES
− Kd[M2+
]
= 0
■
total
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In eq 1, α1 = P.I./[ZIrdap]total = 1.82 × 1011 M−1 (without metal ions
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K
DOI: 10.1021/acs.inorgchem.5b00967
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