A ratiometric and targetable fluorescent sensor for quantification of mitochondrial zinc ions

Article abstract of DOI:10.1002/chem.201103007

Trapping in mitochondria: A ratiometric fluorescent sensor for the detection of mitochondrial Zn²⁺ was synthesised and evaluated on the basis of inhibition of resonance. The sensor, with good selectivity and high sensitivity, can quantify the f

Full text of DOI:10.1002/chem.201103007

DOI: 10.1002/chem.201103007
A Ratiometric and Targetable Fluorescent Sensor for Quantification of
Mitochondrial Zinc Ions
Lin Xue, Guoping Li, Cailan Yu, and Hua Jiang*[a]
After several decades of development, fluorescent sensors
have been recognised as indispensable and efficient molecu-
lar tools that can help monitor and visualise cations or bio-
molecules with high sensitivity and spatial resolution in live
cells or tissues.[1] Undoubtedly, our understanding of metal
ion homeostasis in biology has significantly benefited from
advancements of fluorescent sensors for metal ions. Zn2+
has attracted significant attention because of its critical role
in many biological processes.[2] In spite of worthy attentions
on cytosolic Zn2+, the function of Zn2+ in subcellular com-
partments, such as mitochondria, endoplasmic reticulum,
and Golgi, and underlying dependence between these Zn2+
and cellular processes are still not established well.[3] Hence,
developing targetable fluorescent sensors for monitoring the
zinc level in specific organelles will contribute significantly
to addressing these issues.
To this end, we designed a fluorescent sensor, DQZn2, for
ratiometric detection of mitochondrial Zn2+ (Scheme 1). On
Scheme 1. Structures of DQCd1, DQZn1 and DQZn2.
the basis of our and other observations, 2-picolylamine
(DPA) was employed as an ion chelator that was installed
on the 2-position of quinoline platform so as to achieve high
selectivity for Zn2+ over biological Na+, K+, Ca2+, and
Mg2+ with 1:1 stoichiometry and high affinity with nanomo-
lar or lower Kd values.[8] In the meanwhile, to increase basic-
ity of the sensor, the stronger electron donating nitrogen
atom replaced the oxygen atom on 4-position of quinoline.
We anticipated that this sensor could be protonated under
neutral or even weakly basic media, and consequently
would yield a resonance between quinolinium and onium
resonant structures as observed for DQCd1.[9] The reso-
nance would lead to charge delocalisation, which is more
pronounced in the excited state of the molecule, due to oc-
currence of intramolecular charge transfer (ICT) in polar
media.[10] We envisioned that Zn2+ coordination would
induce the deprotonation of the sensor and consequent in-
hibition of the resonance. The ratiometric measurements
with distinct emission maxima shift can thus be established.
On the other hand, mitochondria are known to be one of
sites that take up Zn2+ in living cells. It is implicated that
the elevation of mitochondrial Zn2+ could lead to intracellu-
Since the fluorescent sensor TSQ was first applied to in
[4]
vitro imaging of Zn2+
,
numerous Zn2+ sensors have been
archived in the past few years. However, only a few small-
molecule fluorescent or genetically encoded zinc sensors
have been designed to target organelles.[3g,5] Moreover, most
of these sensors read out the ion-binding event by emission
intensity changes based on the photoinduced electron trans-
fer (PeT) mechanism. Theoretically, this type of sensor can
provide quantitative measurements of Zn2+, but it is infeasi-
ble to quantify Zn2+ in live cells by these zinc fluorescent
sensors because their emission intensity is significantly influ-
enced by many other factors, such as the sample environ-
ment, sensor concentration, bleaching, and instrumental effi-
ciency. A ratiometric sensor with self-calibration with dual
emission maxima can eliminate most or all ambiguities and
could be an ideal solution for quantitatively measuring in-
tracellular ions.[6] So far, various ratiometric Zn2+-selective
sensors are available,[7] but unfortunately, only several exam-
ples of ratiometric and targetable sensors have been reali-
sed.[3g,5d] Design of targetable and ratiometric sensors for
quantification of Zn2+ in specific organelles is, therefore, im-
portant, yet remains as one of the greatest challenges.
11]
lar H2O2 accumulation and mitochondrial dysfunction.[3c,
Therefore, it is important to quantify Zn2+ levels in mito-
chondria. The triphenylphosphonium salt (TPP), an effective
mitochondrial targeting group, was thus chosen and attached
to the sensor.[12] To minimise the influence of the TPP group
on the photophysical properties of the sensor, it was separat-
ed from the fluorophore by long linkers.[5e] Furthermore,
DQZn1, was also prepared as a control sensor. The synthe-
sis procedures are described in the Supporting Information.
[a] L. Xue, G. Li, C. Yu, Prof. H. Jiang
Beijing National Laboratory for Molecular Sciences
CAS Key Laboratory of Photochemistry, Institute of Chemistry
Chinese Academy of Science
Beijing, 100190 (P.R. China)
E-mail: hjiang@iccas.ac.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103007.
1050
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1050 – 1054

COMMUNICATION
Both sensors had good solubilities (~20 mm, Figure S1 in the
Supporting Information) in aqueous buffer.
cence emission (lem =550 nm, F=0.11) in aqueous buffer.
Although a longer spacer (C6) was chosen to bridge the
TPP and piperidino groups to minimise the effect of TPP on
the properties of the fluorophore, the pKa1 value of DQZn2
was still about 0.4 units lower in comparison with that (8.05)
of its precursor, DQZn1. This is presumably because of the
repulsion effect from the positive charge on the TPP group,
which is unfavourable for protonation and resonant charge
delocalisation.
DQZn2 exhibited fluorescence maxima at 550 nm with
quantum yield of 0.11 in HEPES buffer (10 mm HEPES,
0.1m NaCl, pH 7.4). Upon addition of Zn2+ to the DQZn2
solution, the emission intensity at around 450–560 nm in-
creased significantly, but the emission intensity at about
570–670 nm decreased slightly with an isoemissive point at
567 nm (Figure 1 and Figure S3 in the Supporting Informa-
Firstly, by measuring pH dependence in aqueous buffer
we found that DQZn1 has two pKa values, pKa1 =8.05 and
pKa2 =4.84 (Figure S2 in the Supporting Information). As
expected, the pKa1 value of DQZn1 is obviously larger than
that of DQCd1 (Table 1); this demonstrates that the basicity
Table 1. Spectroscopic properties of the sensors and their zinc complexe-
s.[a]
lem [nm],[b] F[c]
Sensor
pKa1
pKa2
ligand
complex
DQCd1[d]
DQZn1
DQZn2
7.31Æ0.01
8.05Æ0.02
7.66Æ0.02
4.46Æ0.02
4.84Æ0.01
4.57Æ0.02
558, 0.17
550, 0.11
550, 0.11
510, 0.06
507, 0.15
504, 0.22
[a] HEPES (10 mm), NaCl (0.1m), pH 7.4, 258C. [b] The maximum emis-
sion intensity. [c] Quinine sulfate was used as the standard for quantum
yield measurements; [d] see ref. [9].
of DQZn1 is stronger and the nitrogen atom is a better
donor than the oxygen atom to afford the resonance charge
transfer. DQZn2 has similar protonation behaviour to
DQCd1 and DQZn1 with two pKa values of 7.66 and 4.57.
The four nitrogen atoms including the DPA moiety and the
quinolinic nitrogen form a proton binding pocket for the
proton (Scheme 2);[13] this results in a combined pKa1 value
and suppression of the photo-induced electron transfer
(PeT) quenching from the tertiary amine of the DPA
moiety. Therefore, DQZn2 displays considerable fluores-
Figure 1. Fluorescence spectra of 10 mm DQZn2 in the presence of free
Zn2+ (0–45 nm). The free Zn2+ was regulated by using the NTA-Zn2+ (0–
1.8 mm) buffer (10 mm HEPES, 0.1m NaCl, 2 mm NTA, pH 7.4, lex
=
405 nm). Insert: the ratio changes (F504/F550 nm) as a function of Zn2+ con-
centration. The solid lines represent the nonlinear least-squares fits to
the experimental data; NTA: nitrilotriacetic acid.
tion). The blue shift of 46 nm in the emission maxima (from
550 to 504 nm) and twofold enhancement in quantum yield
demonstrates that Zn2+ kicked off the proton at the quino-
linic site by coordination and subsequently inhibited reso-
nance. The ratios of emission intensity (F504/F550 nm) were
found to be 0.39 and 1.94 in the absence and presence of
Zn2+, respectively; this clearly indicates that DQZn2 is an
excellent ratiometric fluorescent sensor for Zn2+ based on
the mechanism of inhibition of resonance. The dissociation
constants (Kd) of DQZn2 were determined by using nonlin-
ear least-squares fit analysis of the ratio of the emission in-
tensity at selected wavelengths in Zn2+-NTA buffered solu-
tions (Figure 1 and Figure S5a in the Supporting Informa-
tion). As expected, DQZn2 displayed a favourable Kd of
0.45
N
Scheme 2. Proposed ratiometric sensing strategy.
Chem. Eur. J. 2012, 18, 1050 – 1054
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1051

H. Jiang et al.
that mitochondria are more basic than the cytosol,[14] the flu-
orimetric titration experiments were repeated in more basic
media (10 mm HEPES, 0.1m NaCl, pH 8.0). As expected,
the photophysical response of DQZn2 to Zn2+ was not seri-
ously disturbed (Figure S4 in the Supporting Information).
The dissociation constant of DQZn2 was also measured in
more basic solutions (pH 8) in the presence of NTA to be
more, the competition experiments indicate that Ca2+, Mg2+
,
and K+ exerted negligible effect on Zn2+ sensing by both in-
tensity and ratio calibration.
Furthermore, we examined the pH-dependence of
DQZn2 and its Zn2+ complex (Zn–DQZn2) through the ra-
tiometric responses (Figure S7 in the Supporting Informa-
tion). In the biological pH range of 5–8, the ratio values of
both DQZn2 and Zn–DQZn2 were almost insensitive. This
indicates that pH changes do not affect the ratiometric
signal output of DQZn2 under physiological conditions.
Before the cellular imaging experiments, we measured the
cytotoxicity of DQZn2 using the CCK-8 assay. The results
demonstrate that DQZn2 is almost nontoxic over a 24 h
period (Table S1 in the Supporting Information) and the
TPP targeting group did not obviously alter the cytotoxicity.
To confirm whether DQZn2 is able to localise to mitochon-
dria, a colocalisation experiment was performed by co-stain-
ing NIH3T3 cells with the commercially available markers
LysoTracker Red DND-99 (Figure S8 in the Supporting In-
formation), which is a lysosomal marker, and MitoTracker
Deep Red (Figure 3b), which is a mitochondrial tracker.
0.15
G
Thus, we believe that DQZn2 can respond to subnanomolar
Zn2+ and monitor the Zn2+ level in mitochondria with suffi-
cient sensitivity.
To study the selectivity of DQZn2 for Zn2+, its fluores-
cent properties in the presence of various cations were ex-
amined in aqueous buffer (Figure 2 and Figure S6 in the
Figure 3. DQZn2 colocalises to mitochondria in live NIH3T3 cells. Cells
were stained with: a) DQZn2 (1.5 mm), and b) MitoTracker Deep Red
(50 nm) for 30 min at 258C in DMEM; c) overlay of a) and b); scale bar:
20 mm.
The fluorescence observed from DQZn2 and MitoTracker
overlapped very well (Figure 3c) with a Pearsonꢁs colocali-
sation coefficient of 0.86, whereas the colocalisation coeffi-
cient between DQZn2 and LysoTracker was only 0.23; this
indicates that DQZn2 is mainly localised to mitochondria in
live cells.
Next, we employed DQZn2 to detect the mitochondrial
Zn2+ levels in living NIH3T3 cells. Incubation (30 min, in
serum-free DMEM) of the cells and subsequent washing
with phosphate buffered saline (PBS) afforded a stained
sample that showed clear fluorescence at dual-emission
channels of 430–490 nm (green) and 520–580 nm (red)
under 405 nm excitation (Figure S9 in the Supporting Infor-
mation). The ratio calculated as Fgreen/Fred gave an average
value of 1.17Æ0.045. Upon treatments with a low dose of
ZnSO4 (25 mm) and 2-mercaptopyridine N-oxide (10 mm) for
2 min, the ratio rapidly increased to 1.59Æ0.093 (P<0.001,
n=5). Sequestration of Zn2+ with the membrane-permeable
Figure 2. a) Fluorescence spectra of DQZn2 (10 mm) in the presence of
various metal ions (10 mm Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+
,
Hg2+, Pb2+, and 1 mm Mg2+, Ca2+, and K+) in buffer solution (10 mm
HEPES, 0.1m NaCl, pH 7.4, lex =405 nm). b) Metal ion selectivity pro-
files of DQZn2 (10 mm). Gray bars represent the relative emission inten-
sity (F/F0, at 504 nm) of DQZn2 in the presence of various metal ions.
Black bars represent the fluorescence intensity of DQZn2 in the pres-
ence of the indicated metal ions, followed by Zn2+ (10 mm).
Supporting Information). As expected, the biologically
abundant metal ions, such as Na+, K+, Ca2+, and Mg2+, did
not produce obvious effect on the fluorescence or ratio cali-
bration of DQZn2 at millimolar concentration (1 mm), while
Fe2+, Co2+, Ni2+ and Cu2+ strongly quenched the fluores-
cence as observed in many other sensory systems. These
free cations would induce no obvious influence in vivo due
to their extremely low concentrations. It should be noted
that Cd2+, which exhibits many properties similar to those
of Zn2+, did not enhance fluorescence (Figure 2a). Further-
chelator
TPEN
(50 mm;
N,N,N’,N’-tetrakis(2-pyridyl-
AHCTUNGTREGmNNUN ethyl)ethylenediamine) rapidly reversed the ratio signals;
this indicates that the increase in ratio depends on mito-
chondrial Zn2+ changes (Figure 4). However, the membrane
1052
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1050 – 1054

Fluorescent Sensor for Mitochondrial Zinc Ions
COMMUNICATION
quent Zn2+ release from these MT to the cytoplasm.[16]
Therefore, we made use of this molecule to elevate the cyto-
solic Zn2+ concentration in NIH3T3 cells. As shown in
Figure 5 and Figure S11 in the Supporting Information, the
Figure 4. Fluorescence ratio images of DQZn2-labelled (1.5 mm) NIH3T3
cells in PBS. a) Image of the emission window Fgreen/Fred; scale bar:
20 mm; ratio bar: 0.2–3.0. b) Cells were treated with ZnSO4 (25 mm) and
2-mercaptopyridine N-oxide (10 mm) for 2 min. c) Cells were treated with
TPEN (50 mm) for 5 min. d) NIH3T3 cells were supplemented with
TPEN (10 mm) in the growth medium for 15 min and incubated with
DQZn2 (1.5 mm) for 30 min. e) Statistical analyses were performed with a
two-tailed Studentꢁs t-test (n=5) relative to the control. Asterisk (*) in-
dicates P<0.01, and error bars are ÆSEM. The ratio (Fgreen/Fred) was cal-
culated as Em430–490/Em520–580 nm upon 405 nm excitation.
potential sensitive dye, Rhodamine 123, did not show statis-
tically significant changes with ZnSO4 and pyrithione treat-
ment; this suggests that the ratio changes did not result
from mitochondrial membrane potential changes (Fig-
ure S10 in the Supporting Information). Moreover, we pre-
treated the cells with TPEN (10 mm) in the growth medium
for 15 min to suppress Zn2+ exchange in the mitochondria
and then incubated them with DQZn2 for 30 min at 258C.
We found that TPEN leads to a significant decrease in the
ratio (r=0.79Æ0.033) in contrast to the case without TPEN;
this indicates that DQZn2 can be used to image Zn2+ fluctu-
ations in mitochondria.
For quantitative analysis of this variable Zn2+ pool, the
intracellular dissociation constant was calibrated according
to a known procedure.[3e,15] The intracellular mitochondrial
Zn2+ level was maintained by using HEPES buffer (20 mm
HEPES, 0.1m NaCl, 1 mm CaEDTA) with known concentra-
tions of free Zn2+. The mean ratio values for each well were
Figure 5. a) Fluorescence ratio images of DQZn2-labelled (1.5 mm)
NIH3T3 cells in PBS, treated with SNOC (100 mm) and further treated
with TPEN (50 mm); scale bar: 20 mm; ratio bar: 0.2–3.0. b) Relative ratio
value of emission intensity as a function of time. c) Statistical analyses
were performed with a two-tailed Studentꢁs t-test (n=5) relative to the
control. Asterisk (*) indicates P<0.05, (**) indicates P<0.01 and error
bars are ÆSEM. The ratio (Fgreen/Fred) was calculated as Em430–490/Em520–
plotted and an apparent Kd of 77
N
from the calibration curve from the microscope by using
nonlinear least-squares fit analysis (Figure S12 in the Sup-
porting Information). The value is in accord with the fitted
upon 405 nm excitation.
580 nm
Kd of 150
N
using the fluorometer. Therefore, this fluctuating mitochon-
dual-emission ratiometric imaging of the cells with DQZn2
showed that the ratio of Fgreen/Fred gradually increased after
treatment with the NO donor S-nitrosocystein (SNOC,
100 mm),[17] and reached 1.64Æ0.10 (P<0.01, n=5) after
10 min. The observed changes in the ratios were immediate-
ly decreased by TPEN treatment. These data demonstrate
that the endogenously released Zn2+ was rapidly taken up
by mitochondria within 1 min (P<0.05, n=5), and establish
that DQZn2 is able to monitor the mitochondrial Zn2+ level
changes through the ratiometric approach. Thus, we believe
drial Zn2+ concentration was calculated to be 72
(n=5) by using the corrected dissociation constant.
Since DQZn2 can monitor intracellular Zn2+ concentra-
tion changes with high sensitivity, we used this chemical tool
to detect the mitochondrial homeostasis in endogenous zinc
release. It is generally acknowledged that nitric oxide is an
important regulator for both physiology and pathology. NO
has been found to induce S-nitrosation and conformational
changes of intracellular metallothionein (MT), and conse-
Chem. Eur. J. 2012, 18, 1050 – 1054
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1053

H. Jiang et al.
[4] C. J. Frederickson, E. J. Kasarskis, D. Ringo, R. E. Frederickson, J.
Neurosci. Methods 1987, 20, 91–103.
that DQZn2 is a prominent sensor for quantifying mito-
chondrial Zn2+ levels in living cells and can be useful for in-
vestigations of mitochondrial Zn2+ biology.
[5] a) S. L. Sensi, D. Ton-That, J. H. Weiss, A. Rothe, K. R. Gee, Cell
Calcium 2003, 34, 281–284; b) E. Tomat, E. M. Nolan, J. Jaworski,
S. J. Lippard, J. Am. Chem. Soc. 2008, 130, 15776–15777; c) J. L.
Vinkenborg, T. J. Nicolson, E. A. Bellomo, M. S. Koay, G. A. Rutter,
M. Merkx, Nat. Methods 2009, 6, 737–740; d) Y. Qin, P. J. Dittmer,
J. G. Park, K. B. Jansen, A. E. Palmer, Proc. Natl. Acad. Sci. USA
2011, 108, 7351–7356; e) G. Masanta, C. S. Lim, H. J. Kim, J. H.
Han, H. M. Kim, B. R. Cho, J. Am. Chem. Soc. 2011, 133, 5698–
5700.
In summary, we present a quinoline-based ratiometric
sensor, DQZn2, according to the principle of cation-induced
inhibition of resonance. It allows single-excitation, dual-
emission detection of Zn2+ with significant blue shift of
46 nm in emission and remarkable changes in the ratio of
fiveACHTUNGTRENNUNG
fold in response to Zn2+ with subnanomolar Kd, under
[6] K. W. Dunn, S. Mayor, J. N. Myers, F. R. Maxfield, FASEB J. 1994,
8, 573–582.
physiological conditions. Moreover, we emphasise that
DQZn2, which is attached with the TPP targeting group,
can localised to the mitochondria in living cells and can
indeed be used to visualise the changes of mitochondrial
Zn2+ level. Furthermore, we took advantage of DQZn2-
stained ratiometric approach for quantification of the free
mitochondrial Zn2+ concentration in NIH3T3 cells. This mo-
lecular tool should be useful for investigations of mitochon-
drial Zn2+ biology. The design strategy presented here is ra-
tional and practical for developing new ratiometric sensors
for other biologically relevant analytes in subcellular lo-
cales; this is currently in progress.
[7] Selected examples: a) P. Carol, S. Sreejith, A. Ajayaghosh, Chem.
Asian J. 2007, 2, 338–348; b) J. L. Vinkenborg, M. S. Koay, M.
Merkx, Curr. Opin. Chem. Biol. 2010, 14, 231–237; c) M. D. Pluth,
E. Tomat, S. J. Lippard, Annu Rev Biochem. 2011, 80, 333–355.
[8] a) L. Xue, H. H. Wang, X. J. Wang, H. Jiang, Inorg. Chem. 2008, 47,
4310–4318; b) L. Xue, C. Liu, H. Jiang, Chem. Commun. 2009,
1061–1063; c) K. Hanaoka, K. Kikuchi, H. Kojima, Y. Urano, T.
Nagano, J. Am. Chem. Soc. 2004, 126, 12470–12476; d) X. Y. Chen,
J. Shi, Y. M. Li, F. L. Wang, X. Wu, Q. X. Guo, L. Liu, Org. Lett.
2009, 11, 4426–4429.
[9] L. Xue, G. P. Li, Q. Liu, H. H. Wang, C. Liu, X. L. Ding, S. G. He,
H. Jiang, Inorg. Chem. 2011, 50, 3680–3690.
[10] a) Z. Dega-Szafran, A. Kania, B. Nowak-Wydra, M. Szafran, J. Mol.
Struct. 1994, 322, 223–232; b) P. Chou, C. Wei, J. Phys. Chem. 1996,
100, 17059–17066; c) D. Panda, D. Ghosh, A. Datta, J. Photochem.
Photobiol. A 2009, 207, 254–259.
Acknowledgements
[11] a) S. L. Sensi, H. Z. Yin, S. G. Carriedo, S. S. Rao, J. H. Weiss, Proc.
Natl. Acad. Sci. USA 1999, 96, 2414–2419; b) S. L. Sensi, H. Z. Yin,
J. H. Weiss, Eur. J. Neurosci. 2000, 12, 3813–3818; c) S. L. Sensi, P.
Paoletti, A. I. Bush, I. Sekler, Nat. Rev. Neurosci. 2009, 10, 780–791.
[12] a) B. C. Dickinson, C. J. Chang, J. Am. Chem. Soc. 2008, 130, 9638–
9639; b) M. F. Ross, T. A. Prime, I. Abakumova, A. M. James, C. M.
Porteous, R. A. J. Smith, M. P. Murphy, Biochem. J. 2008, 411, 633–
645; c) B. C. Dickinson, D. Srikun, C. J. Chang, Curr. Opin. Chem.
Biol. 2010, 14, 50–56; d) S. C. Dodani, S. C. Leary, P. A. Cobine,
D. R. Winge, C. J. Chang, J. Am. Chem. Soc. 2011, 133, 8606–8616.
[13] a) B. A. Wong, S. Friedle, S. J. Lippard, J. Am. Chem. Soc. 2009, 131,
7142–7152; b) D. Buccella, J. A. Horowitz, S. J. Lippard, J. Am.
Chem. Soc. 2011, 133, 4101–4114.
We thank the National Natural Science Foundation of China (Nos.
90813002, 21102148, 21125205), National Basic Research Program of
China (No. 2011CB935800), the “100 Talent” program of the Chinese
Academy of Sciences, and the State Key Laboratory of Fine Chemicals,
Department of Chemical Engineering, Dalian University of Technology
(KF1001) for financial supports.
Keywords: biosensors · fluorescent probes · mitochondria ·
quinoline · zinc ions
[14] a) J. Llopis, J. M. McCaffery, A. Miyawaki, M. Farquhar, R. Y.
Tsien, Proc. Natl. Acad. Sci. USA 1998, 95, 6803–6808; b) M.
Nakano, H. Imamura, T. Nagai, H. Noji, ACS Chem. Biol. 2011, 6,
709–715.
[1] a) D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4,
168–175; b) T. Terai, T. Nagano, Curr. Opin. Chem. Biol. 2008, 12,
515–521.
[15] a) C. Cheng, I. J. Reynolds, J. Neurochem. 2002, 71, 2401–2410;
b) S. L. Sensi, D. Ton-That, J. H. Weiss, Neurobiol. Dis. 2002, 10,
100–108.
[16] L. L. Pearce, R. E. Gandley, W. Han, K. Wasserloos, M. Stitt, A. J.
Kanai, M. K. McLaughlin, B. R. Pitt, E. S. Levitan, Proc. Natl. Acad.
Sci. USA 2000, 97, 477–482.
[2] a) X. Xie, T. G. Smart, Nature 1991, 349, 521–524; b) J. M. Berg, Y.
Shi, Science 1996, 271, 1081–1085.
[3] a) R. D. Palmiter, S. D. Findley, EMBO J. 1995, 14, 639–649;
b) S. C. Burdette, S. J. Lippard, Proc. Natl. Acad. Sci. USA 2003,
100, 3605–3610; c) S. L. Sensi, D. Ton-That, P. G. Sullivan, E. A.
Jonas, K. R. Gee, L. K. Kaczmarek, J. H. Weiss, Proc. Natl. Acad.
Sci. USA 2003, 100, 6157–6162; d) D. J. Eide, Biochim. Biophys.
Acta. 2006, 1763, 711–722; e) R. A. Bozym, R. B. Thompson, A. K.
Stoddard, C. A. Fierke, ACS Chem. Biol. 2006, 1, 103–111; f) A.
Krꢂtzel, W. Maret, J. Biol. Inorg. Chem. 2006, 11, 1049–1062; g) P. J.
Dittmer, J. G. Miranda, J. A. Gorski, A. E. Palmer, J Biol. Chem.
2009, 284, 16289–16297.
[17] K. D. Krçncke, V. Kolb-Bachofen, Methods Enzymol. 1999, 301,
126–135.
Received: September 25, 2011
Published online: December 21, 2011
1054
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 1050 – 1054