A FRET-based indicator for imaging mitochondrial zinc ions

Article abstract of DOI:10.1039/c1cc14580k

A strategy based on fluorescence resonance energy transfer (FRET) to transform a red-emitting fluorophore into a ratiometric indicator for mitochondrial Zn^II is demonstrated.

Full text of DOI:10.1039/c1cc14580k

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 11730–11732
www.rsc.org/chemcomm
COMMUNICATION
A FRET-based indicator for imaging mitochondrial zinc ionsw
Kesavapillai Sreenath,a John R. Allen,b Michael W. Davidson*b and Lei Zhu*a
Received 27th July 2011, Accepted 30th August 2011
DOI: 10.1039/c1cc14580k
A strategy based on fluorescence resonance energy transfer
(FRET) to transform a red-emitting fluorophore into a ratio-
metric indicator for mitochondrial ZnII is demonstrated.
(2) Fluorescent indicators with red emission (lem close to or
4 600 nm) are desirable partly because their spectral windows
lie outside the range of autofluorescence of biological specimens.
The development of red-emitting indicators for zinc(II) has
been based on the functionalization of a fluorophore structure
with a zinc(II) coordinating ligand.22–28 This practice has
largely been a trial-and-error process in which the effects of
functionalization on the photophysical properties of the fluoro-
phore are hardly predictable. In this work, we present a general
strategy to transform a red fluorophore into an indicator that
targets mitochondrial zinc(II).
The proper functions of zinc(II)-dependent biomolecules are
sensitively affected by the availability of zinc(II).1–4 Therefore,
homeostasis of zinc(II)–the ability to adjust zinc(II) distribution
and to mediate zinc(II) transport on demand–is critical in
maintaining the well-being of organisms.5 The disruption of
zinc(II) homeostasis leads to diseases,6 ranging from develop-
mental disorders to neurodegenerative diseases.7
The designed molecule consists of a FRET donor–acceptor
pair and a mitochondrial targeting moiety (Fig. 1). The FRET
donor is a charge-transfer type fluorophore which binds
zinc(II) to result in a bathochromic shift of emission. The
spectral overlap between donor and acceptor grows upon
zinc(II) coordination, thus enhancing the efficiency of FRET.
To deliver the probe selectively to mitochondria, a lipophilic
triphenylphosphonium (TPP) moiety, which is a known
mitochondrial targeting functionality,29 is attached through
an alkyl chain to the indicator. A handful of mitochondrial
targeting fluorescent indicators have been constructed using
the TPP-tagging strategy,30 including a case of indicator for
zinc(II) by Cho et al. which was published while this manu-
script was in preparation.18
Motivated by the challenge of determining the distribution
and dynamics of zinc(II) in living specimens, significant efforts
have been put forth to quantify and to image free, or mobile
zinc(II)8 in vivo and in vitro.9–11 Fluorescence-based methods
have received considerable attention due to its adequately high
spatial and temporal resolutions and the rapidly growing
sophistication and accessibility of fluorescence micro-
scopes.12,13 To complement fluorescence microscopy, fluores-
cent indicators for zinc(II) are created based on innovative
applications of metal coordination-dependent excited state
relaxation processes of fluorophores.14 Various excited state
occurrences, such as internal charge transfer, photo-induced
electron transfer, excited state proton transfer, and fluores-
cence resonance energy transfer (FRET), have been integrated
into fluorescent indicators targeting zinc(II).15
In this work, 5-(4-methoxystyryl)-50-methyl-2,20-bipyridine
(1)31 and diamino-substituted naphthalenediimide (NDI, red
in Fig. 2)32 were selected as the FRET donor and acceptor,
respectively (Fig. 2). The charge-transfer excited state of
The remaining challenges in the area of zinc(II) indicator
development include the following two issues that this work
aims to address. (1) High spatial resolution is one of the most
distinctive advantages of fluorescence microscopy over other
imaging methods.10,16 Therefore, fluorescent indicators with
defined subcellular localization properties would maximize the
impact of fluorescence microscopy in imaging applications.
Thus far, only a few synthetic indicators are reported to target
organellar zinc(II).17,18 Genetically encoded indicators for
organellar zinc(II) based on a similar strategy for engineering
calcium(II) sensor Cameleon have also appeared.19–21
a Department of Chemistry and Biochemistry, Florida State
University, Tallahassee, FL 32306-4390, USA.
E-mail: lzhu@chem.fsu.edu
b National High Magnetic Field Laboratory and Department of
Biological Science, Florida State University, 1800 East Paul Dirac
Drive, Tallahassee, FL 32310, USA.
E-mail: davidson@magnet.fsu.edu
w Electronic supplementary information (ESI) available: Experimental
procedures and additional spectra. See DOI: 10.1039/c1cc14580k
Fig. 1 Strategy for transforming a red fluorophore (red rectangle)
into a zinc(II) indictor via zinc(II)-induced FRET. Blue and green
circles represent free and bound FRET-donor. Blue and red arrows
represent blue excitation and red emission, respectively. Diamond
‘‘+’’ on the right: a lipophilic mitochondrial-targeting cation.
c
11730 Chem. Commun., 2011, 47, 11730–11732
This journal is The Royal Society of Chemistry 2011

View Online
emission at 630 nm intensifies, and reaches saturation with
12-fold enhancement in integrated intensity (see Table S1 of the
ESI for spectroscopic dataw). However, when NDI is directly
excited at 600 nm, the intensity is not affected much by the
variation of zinc(II) concentration ([Zn2+]) (Fig. S3, ESIw).
These observations are consistent with the involvement of a
zinc(II)-modulated intramolecular excitation energy transfer
process. Furthermore, the differed sensitivity to excitation
wavelength of 4 offers an opportunity to correlate [Zn2+
]
and fluorescence change ratiometrically.35 As shown in Fig.
S4, ESI,w the ratio of integrated fluorescence upon excitation
at 400 and 600 nm is sensitively dependent on [Zn2+]. This
correlation is independent of the concentration of indicator 4
in the linear portion of the calibration curve.
The sensitized excitation of the NDI moiety in 4 when
excited at 400 nm is supported by the observation of the
following control experiment. ZnCl2 was titrated into a mixture
of compounds 1 and 2 of equal concentration (2.5 mM) while
the emission spectra were collected upon excitation at 400 nm.
Only a weak band of NDI was seen at the beginning of the
titration which was quickly overwhelmed by the growing green
emission from [Zn(1)]2+ (Fig. S5, ESIw). In compound 4
however, the excitation energy of the zinc(II)-bound donor
portion, which is equivalent to [Zn(1)]2+, is captured by the
NDI acceptor, which emits instead to result in the observations
in Fig. 3b.
Fig. 2 Structures of zinc(II) indicators (4, 5) and model systems (1–3).
compound 1 can be stabilized via metal coordination at the
bipy site.31,33 Consequently, upon addition of zinc(II) to a
solution of 1, its emission spectrum undergoes a bathochromic
shift (from blue to green in Fig. S1, ESIw) to enable a
significant spectral overlap between the emission of zinc(II)-
bound 1 and the S0-S1 absorption band of NDI dye 2
(Fig. S1, ESIw). NDI was chosen as the acceptor due to its
synthetic versatility, where each of the four sides of NDI can
be functionalized individually, and its tunable fluorescence
properties.34 When 1 and 2 are covalently assembled to afford
compound 4, the efficiency of intramolecular FRET in 4 is
expected to be a function of zinc(II) concentration.
The fluorescence dependence of 4 on the identity of metal
ion was evaluated (Fig. S7, ESIw). The alkali and alkaline
earth metal ions that are abundant in biological systems do
not elicit a fluorescence response of 4. Although manganese(II)
and lead(II) are known to bind bipy, they fail to alter the
fluorescence of 4. Not surprisingly, cadmium(II) acts similarly
to zinc(II) in resulting strong fluorescence from NDI, whereas
iron(II) and copper(II) quench fluorescence. The observed
metal ion selectivity can be understood based on the Irvin-
Williams series and HSAB theory. Although cadmium(II),
iron(II), and copper(II) are potential interfering ions, the
applicability of 4 in microscopic imaging experiments shall
not be significantly compromised due to the low abundances
of these ions in biological specimens.
The syntheses of the separate FRET donor and acceptor
systems (1–3) and indicators for zinc(II) (4, 5) are described in
the ESI.w Absorption spectrum of indicator 4 in CH3CN
consists of two bands over 300 nm (Fig. 3a, blue trace). The
band centered at 346 nm corresponds to the absorption of the
bipy-containing FRET donor moiety, which we term the D
band. The band centered at 600 nm corresponds to the NDI
moiety which is termed the A band. Upon addition of increasing
amount of ZnCl2, as a result of zinc(II) coordination at the bipy
moiety, the charge-transfer D band undergoes a bathochromic
shift from 346 nm to 370 nm. The A band, meanwhile,
experiences a slight hypochromic shift.
The titration experiments using indicator 4 were also carried
out in an aqueous/organic mixed solvent (1 : 9 MOPS buffer
(pH 7.2)/CH3CN). Compound 4 shows a 3-fold fluorescence
enhancement on increasing [Zn2+] (Fig. S9, ESIw). Compared
to the observations in CH3CN, the fluorescence is rather dim.
The loss of fluorescence can be explained by the hydrogen
bonding interaction between the protic solvent and the carbonyl
groups on NDI, which provides a non-radiative deactivation
pathway.32
The fluorescence titration profile of 4 with ZnCl2 upon
excitation at 400 nm is shown in Fig. 3b. The spectrum of 4
exhibits a weak emission at 630 nm. This signal may have
arisen from the direct excitation of the NDI component. Upon
addition of increasing amount of ZnCl2 from 0–18 mM, the
The pKa of compound 1, which is also the ionophore of
compound 4, is 4.6. The intensity of 1 exhibits little change in
the range of pH 6.0–9.0 (Fig. S10, ESIw). By extension, the
fluorescence of 4 and 5 would not be affected by the fluctuations
of pH within the physiological range.
To equip the indicator with mitochondrial targeting capability,
we installed the triphenylphosphonium (TPP) group on 2 and
4 to afford indicators 3 and 5, respectively. The colocalization
properties of red-emitting 2–5 were studied using HeLa (S3)
Fig. 3 (a) Absorption spectra for the titration of 4 (3.0 mM) with
ZnCl2 (0–18 mM) in CH3CN. (b) Fluorescence spectral change of
4 (3.0 mM) with different concentrations of ZnCl2 (0–18 mM) in CH3CN
(lex 400 nm).
cells transfected with mCerulean3 TOMM-20,
a cyan
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 11730–11732 11731

View Online
compound 5 offers attractive features including a large spectral
separation between excitation and emission, little pH sensitivity
within the physiological window, ratiometric imaging capability,
and defined subcellular localization preference. The ongoing
work includes the application of the reported FRET strategy
to convert other red-emitting fluorophores into ion indicators,
as well as strategy development for targeting other subcellular
organelles.
Notes and references
1 S. J. Lippard and J. M. Berg, Principles of Bioinorganic Chemistry,
University Science Books, Mill Valley, CA, 1994.
2 B. L. Vallee and K. H. Falchuk, Psychological Reports, 1993, 73, 79.
3 J. M. Berg and Y. Shi, Science, 1996, 271, 1081.
Fig. 4 Confocal fluorescence images of living HeLa (S3) cells transfected
with mCerulean3 TOMM-20 that were incubated with 5 (1.8 mM,
A–C) and 4 (6.0 mM, D–F) for 30 min. Left: green channel, lex 405 nm,
lem 425–475 nm; middle: red channel, lex 543 nm, lem 580–680 nm;
right: merged image. Scale bar: 10 mm.
4 W. N. Lipscomb and N. Strater, Chem. Rev., 1996, 96, 2375.
¨
5 W. Maret, BioMetals, 2001, 14, 187.
6 J. Nutr., 2000, 130, 14715S–1483S.
7 C. J. Frederickson, J.-Y. Koh and A. I. Bush, Nat. Rev. Neurosci.,
2005, 6, 449.
fluorescent protein (CFP) fused with a mitochondrial targeting
sequence whose emission is shown in the green channel
(Fig. 4A and D). Lacking the TPP group, compound 4 forms
particulates intracellularly (Fig. 4E) which show poor coloca-
lization with the CFP (Fig. 4F). The overall intracellular
fluorescence intensity from compound 4 is low, which is
consistent with the observed weak fluorescence of the NDI
fluorophore in aqueous solutions. The TPP-containing 5
(Fig. 4B), on the other hand, localizes selectively in mitochondria
(Fig. 4C) and displays a surprisingly strong intracellular
fluorescence relative to that of 4. Similar contrast was observed
between the model compounds 2 and 3 (Fig. S15, ESIw). The
strong fluorescence of intracellular 5 may be attributed to the
dispersion of the indicator molecules into the inner mitochondrial
membrane29 or a relatively organic-like mitochondrial matrix.
Further investigation to account for this observation is
ongoing.
8 A. Kre˛zel and W. Maret, JBIC, J. Biol. Inorg. Chem., 2006,
˙
11, 1049.
9 E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008,
108, 1517.
10 R. McRae, P. Bagchi, S. Sumalekshmy and C. J. Fahrni, Chem.
Rev., 2009, 109, 4780.
11 E. Tomat and S. J. Lippard, Curr. Opin. Chem. Biol., 2010, 14, 225.
12 E. M. Nolan and S. J. Lippard, Acc. Chem. Res., 2009, 42, 193.
13 Z. Xu, J. Yoon and D. R. Spring, Chem. Soc. Rev., 2010, 39, 1996.
14 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem.
Rev., 1997, 97, 1515.
15 C. J. Chang and S. J. Lippard, Met. Ions Life Sci., 2006, 1, 321.
16 M. Fernandez-Suarez and A. Y. Ting, Nat. Rev. Mol. Cell Biol.,
´ ´
2008, 9, 929.
17 E. Tomat, E. M. Nolan, J. Jaworski and S. J. Lippard, J. Am.
Chem. Soc., 2008, 130, 15776.
18 G. Masanta, C. S. Lim, H. J. Kim, J. H. Han, H. M. Kim and
B. R. Cho, J. Am. Chem. Soc., 2011, 133, 5698.
19 P. J. Dittmer, J. G. Miranda, J. A. Gorski and A. E. Palmer,
J. Biol. Chem., 2009, 284, 16289.
20 J. L. Vinkenborg, T. J. Nicolson, E. A. Bellomo, M. S. Koay,
G. A. Rutter and M. Merkx, Nat. Methods, 2009, 6, 737.
21 Y. Qin, P. J. Dittmer, J. G. Park, K. B. Jansen and A. E. Palmer,
Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 7351.
22 S. L. Sensi, D. Ton-That, J. H. Weiss, A. Rothe and K. R. Gee,
Cell Calcium, 2003, 34, 281.
The ability of indicator 5 to report mitochondrial zinc(II)
variation is demonstrated in Fig. 5. Upon excited at 405 nm,
the addition of supplemental zinc(II) in the growth media
(DMEM) leads to an increase in intramolecular fluorescence
of 5 that largely localizes in mitochondria. The addition of
compound 5 to the group of few synthetic indicators that
target mitochondrial zinc(II)17,18,22 shall aid the characterization
of mitochondrial zinc(II) which is intimately correlated to the
chemistry of reactive oxygen species (ROS).36
23 K. Kiyose, H. Kojima, Y. Urano and T. Nagano, J. Am. Chem.
Soc., 2006, 128, 6548.
24 B. Tang, H. Huang, K. Xu, L. Tong, G. Yang, X. Liu and L. An,
Chem. Commun., 2006, 3609–3611.
25 S. Atilgan, T. Ozdemir and E. U. Akkaya, Org. Lett., 2008,
10, 4065.
26 L. Xue, C. Liu and H. Jiang, Chem. Commun., 2009, 1061.
27 P. Du and S. J. Lippard, Inorg. Chem., 2010, 49, 10753.
28 X. Lu, W. Zhu, Y. Xie, X. Li, Y. Gao, F. Li and H. Tian,
Chem.–Eur. J., 2010, 16, 8355.
29 M. P. Murphy and R. A. J. Smith, Annu. Rev. Pharmacol., 2007, 47, 629.
30 B. C. Dickinson and C. J. Chang, J. Am. Chem. Soc., 2008,
130, 9638.
In summary, the conversion of a red-emitting fluorophore
to a zinc(II)-responding indicator using a FRET-based strategy
is demonstrated. The diamino-substituted NDI dye, which has
not drawn much attention in intracellular applications,28 performs
well as the fluorophore in indicator 5. In addition to red-emitting,
31 A. H. Younes, L. Zhang, R. J. Clark and L. Zhu, J. Org. Chem.,
2009, 74, 8761.
32 F. Wurthner, S. Ahmed, C. Thalacker and T. Debaerdemaeker,
¨
Chem.–Eur. J., 2002, 8, 4742.
33 R. J. Wandell, A. H. Younes and L. Zhu, New J. Chem., 2010,
34, 2176.
34 C. Thalacker, C. Roger and F. Wurthner, J. Org. Chem., 2006,
¨
¨
71, 8098.
35 P. Carol, S. Sreejith and A. Ajayaghosh, Chem.–Asian J., 2007,
2, 338.
36 S. L. Sensi, D. Ton-That, P. G. Sullivan, E. A. Jonas, K. R. Gee,
L. K. Kaczmarek and J. H. Weiss, Proc. Natl. Acad. Sci. U. S. A.,
2003, 100, 6157.
Fig. 5 Fluorescence images of HeLa (S3) cells incubated with compound
5 (1.8 mM). (A) No zinc(II) added, and (B) in the presence of 50 mM ZnCl2.
A 405 nm diode laser line was used for excitation. lem 580–680 nm. Scale
bar: 10 mm. The DIC images are included in Fig. S16, ESI.w
c
11732 Chem. Commun., 2011, 47, 11730–11732
This journal is The Royal Society of Chemistry 2011