A fluorescent heteroditopic ligand responding to free zinc ion over six orders of magnitude concentration range

Article abstract of DOI:10.1039/b918729d

A fluorescent heteroditopic ligand useful in live-cell imaging studies responds to free zinc ion concentration over a range of six orders of magnitude in a buffered aqueous solution via dual-channel fluorescence.

Full text of DOI:10.1039/b918729d

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A fluorescent heteroditopic ligand responding to free zinc ion over six
orders of magnitude concentration rangew
Lu Zhang,a Christopher S. Murphy,b Gui-Chao Kuang,a Kristin L. Hazelwood,b
Manuel H. Constantino,a Michael W. Davidson*b and Lei Zhu*a
Received (in Austin, TX, USA) 9th September 2009, Accepted 16th October 2009
First published as an Advance Article on the web 4th November 2009
DOI: 10.1039/b918729d
A fluorescent heteroditopic ligand useful in live-cell imaging
studies responds to free zinc ion concentration over a range of
six orders of magnitude in a buffered aqueous solution via
dual-channel fluorescence.
the cellular uptake efficiencies of various probes may differ,
which complicates quantitative measurements. Individual
probes that are capable of zinc quantification over large
concentration ranges will be far superior. The demand for
such probes is underscored by the unique challenges in zinc
neurobiology. For example, presynaptic release of zinc in
neurons at high or low concentrations has been postulated
to induce the activities of either high-affinity voltage independent
(HAVI) or low-affinity voltage dependent (LAVD) zinc
receptors at the postsynaptic density (PSD).19 Therefore, the
quantitative information of zinc fluctuation during a brain
neuronal event could be best obtained by using a single probe
applicable over a broad concentration range. The zinc-release
data can be correlated to the expression of HAVI or LAVD
receptors which may prove pivotal in unraveling the zinc-
involved signal transduction pathways in brain neurobiology.
To design a probe with a broad concentration coverage, we
first simulated the fluorescence responses to [Zn]f of probes
containing single zinc-binding sites. The effective concentration
range of a ‘‘monotopic’’ probe is B3 orders of magnitude,
inadequate for covering the entire concentration range of
biological free zinc (Fig. S1w). On the other hand, if the
responses from two monotopic probes with an affinity difference
of B103 were combined, an expanded range of 6 orders of
magnitude could be achieved. When the signal of the high-
affinity probe is close to saturation, the low-affinity probe
begins to operate. Therefore, by including two different zinc
coordination sites in one molecule and translating the
information of species distribution into fluorescence, fluorescent
zinc probes with six orders of magnitude concentration coverage
are attainable.
Our interest in zinc coordination chemistry and sensing20,21
led to a fluorescent heteroditopic ligand platform (Fig. 1)
capable of correlating three coordination states (non-, mono-,
and di-coordinated) to three fluorescence states (OFF, ON at
one color, ON at a different color) in acetonitrile.22–25 This
structural framework is ideal for developing zinc probes
effective over large concentration ranges. The ligand is
designed to have a low fluorescence quantum yield (jf) due
to intramolecular photoinduced electron transfer (PET)26–28
from Q (the Quencher, also the high-affinity zinc binding
group) to the excited F (the Fluorophore). In the presence of
zinc, the ion preferentially binds with the high-affinity site Q to
result in fluorescence enhancement at l1.26 The intensity at l1
can be used to correlate zinc concentration values at the low
end. When zinc concentration is high enough to bind with
the second, low-affinity site within F, stabilization of the
The biological functions of zinc ion are multifaceted:1,2 it
(a) serves as a catalytic cofactor for many metalloenzymes,
(b) supports functional tertiary structures of various proteins,
and (c) mediates neuronal signal transduction events.3
Consequently, the fluctuation of free (available for protein
binding)4,5 zinc ion concentration ([Zn]f) may profoundly
affect the physiological state of an organism. Three classes
of proteins: transporters, sensors, and metallothioneins, work
in concert in regulating zinc homeostasis.6 Their activities are
dependent on the type and developmental stage of cells. Such
dependence leads to the varied availability (or concentration)
of free zinc across an organism.
The mechanistic details of zinc homeostasis are not entirely
clear,7,8 partly due to the difficulty in directly mapping the
distribution and tracking the movement of the spectro-
scopically inert free zinc ions. Therefore, zinc ion-selective
fluorescent probes have been the primary tools for the detection
and quantification of biological free zinc. The past decade has
witnessed the rapid progress in the development of zinc
probes,9–13 which have enabled significant advances in the cell
biology of zinc.3,11
Despite the advances, new challenges continue to emerge.
For instance, it is known that the concentrations of free zinc
range from sub-nanomolar5 to almost millimolar14 depending
on cell and organelle types. Quantification over the entire
physiological range would require probes whose fluorescent
signals do not saturate over a concentration span ranging six
orders of magnitude. Some attempts have been made to
address this issue, for example, by using a collection of probes
with different detection ranges.15–18 However, in addition to
being labor-intensive because parallel experiments are needed,
a Department of Chemistry and Biochemistry, Florida State
University, Tallahassee, FL 32306-4390, USA.
E-mail: lzhu@chem.fsu.edu; Fax: +1 850 644 8281;
Tel: +1 850 645 6813
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;
Fax: +1 850 644 8920; Tel: +1 850 644 0542
w Electronic supplementary information (ESI) available: Procedures
for syntheses, absorption and emission titration studies, and live-cell
imaging experiments. Additional spectra, DIC images, and
co-localization scatterplots were included. See DOI: 10.1039/b918729d
ꢀc
This journal is The Royal Society of Chemistry 2009
7408 | Chem. Commun., 2009, 7408–7410

View Article Online
Fig. 2 Fluorescence (A, lex = 340 nm) of 1 (2 mM) at various free
zinc concentrations ([Zn]f) in 10% DMSO aqueous solution (HEPES:
50 mM, pH = 7.0, KNO3: 100 mM; EDTA: 5 mM; HEDTA: 5 mM;
EGTA: 5 mM; NTA: 5 mM). The blue and red arrows represent
spectral changes when [Zn]f is low and high, respectively. The jf values
of three coordination states are listed. (B) The fluorescence intensity at
409 nm and 480 nm, respectively. The double-headed arrows represent
the concentration ranges covered by the two wavelength channels.
Fig. 1 Left: working principle of a fluorescent heteroditopic ligand.
Details in the text. Q: Quencher; F: Fluorophore; CHEF: CHelation-
Enhanced Fluorescence; ICT: Internal (or Intramolecular) Charge
Transfer. Right: structure of compound 1.
charge-transfer (CT) excited state upon coordination results in
an emission bathochromic shift from l1 to l2.29 Therefore, the
intensity at l2, or the intensity ratio at l2 and l1, can be used to
relate zinc concentration at the high end.
30 min. Weak fluorescence was observed without addition of
zinc using the Q-Max Blue filter set (Omega Optical). The
background fluorescence (Fig. 3B) was primarily a result from
the zinc content in the culture media. It was mostly eliminated
after the addition of the membrane-permeable, high-affinity
zinc chelator TPEN (Fig. 3A). Upon increasing zinc
concentration up to 100 mM, the fluorescence intensity rises
as observed in the window of 420 nm to 480 nm (Fig. 3). The
overall fluorescence enhancement at this particular excitation/
emission profile was anticipated based on the solution absorption
and emission titration studies (Fig. 2 and S5w). 1 and its zinc
complex do not appear toxic during the experiment based on
the lack of change in cell morphology.
Heteroditopic ligand 1 reported herein is shown in Fig. 1.
The high-affinity site is the N,N,N0-tris(pyridylmethyl)-ethylene-
amino group, which has been extensively used for zinc
binding.30 Other studies showed that this polyaza moiety is
selective for zinc over calcium and magnesium, the two metals
that might interfere with fluorescence imaging of cellular zinc,
by 8–10 orders of magnitude in affinity.30,31 The low-affinity
site is the 2,20-bipyridyl (bipy) site, which is part of the
fluorophore. The synthesis of 1 is described in the ESIw.
Similar to the reported model system,22 1 shows fluorescence
enhancement followed by an emission bathochromic shift
upon zinc addition in acetonitrile (ESIw). The following
discussion pertains to studies in aqueous solutions.
Similar to the eukaryotic cellular environments,5,32 a zinc
ion buffer system was used in the titration experiments where
the total zinc ion concentrations were high and the free zinc
concentrations were controlled by metal ion chelators EDTA,
HEDTA, EGTA, and NTA. The absorption spectrum of 1
undergoes an overall bathochromic shift upon addition of zinc
(Fig. S5w) due to the eventual coordination at the bipy site to
result in the stabilization of the ICT excited state. Similar to
the observations in acetonitrile, the fluorescence undergoes an
enhancement prior to a bathochromic shift (Fig. 2A). The
sequential coordination at the high- and low-affinity binding
sites results in CHEF and stabilization of the ICT excited
state, respectively. The fluorescence quantum yields (jf) of
various species are listed in Fig. 2A.
The subcellular localization properties of 1 were studied by
co-staining experiments using organelle-specific probes. 1 and
its zinc complex localize in mitochondria as indicated by high
Pearson’s coefficients (PC, 0.86 and 0.90, respectively, Fig. 4D)
determined by co-localization scatterplots (Fig. S7w).33
Mitochondrial zinc can be used for assembling zinc metallo-
enzymes. However, the complete functional profile of
the mitochondrial zinc remains largely uncharacterized.34
The correlation of fluorescence intensity of 1 to [Zn]f at two
wavelengths, which represent the short and long emission
bands, reveals the concentration range of free zinc that 1 can
cover for quantitative analysis (Fig. 2B). The fluorescence
intensity at the short wavelength channel (409 nm) is sensitive
to the change of [Zn]f from 10ꢁ14 M to 10ꢁ11 M; whereas
the intensity at the long wavelength channel (480 nm) can
be related to [Zn]f from 10ꢁ11 M to 10ꢁ8 M. As suggested by
Fig. S1,w by combining the coverage of both channels, the
overall [Zn]f coverage of 1 spans from 10ꢁ14 M to 10ꢁ8 M,
a range of six orders of magnitude.
Fig. 3 (A) to (F): fluorescence images of live HeLa cells (37 1C, 5%
CO2) with added [ZnCl2] at 0 (with 20 mM TPEN), 0, 5 mM, 10 mM,
50 mM, and 100 mM, respectively (DIC images in ESIw). The cells were
incubated with 1 (10 mM) and MitoTracker Red (0.3 mM) for 30 min
before media was replaced. The cells were then incubated with ZnCl2/
pyrithione solutions ([ZnCl2] from 0 to 100 mM; [pyrithione] = 20 mM)
for 10 min. The fluorescence images were acquired using a Q-Imaging
Retiga camera and an Omega Q-Max Blue filter set (excitation 355–405 nm;
emission 420–480 nm) with 500 ms exposure time. The focus was
adjusted in the red channel (MitoTracker Red). Scale bar: 10 mm.
The applicability of 1 in live-cell imaging was evaluated.
HeLa cells (CCL2 line) absorb 1 readily upon incubation up to
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 7408–7410 | 7409

View Article Online
This work was supported by FSU through a start-up fund, a
New Investigator Research (NIR) grant (08KN-16) from the
James
& Esther King Biomedical Research Program
administered by FL DOH, and NSF (CHE 0809201).
Notes and references
1 B. L. Vallee and K. H. Falchuk, Physiol. Rev., 1993, 73, 79.
2 J. M. Berg and Y. Shi, Science, 1996, 271, 1081.
3 C. J. Frederickson, J.-Y. Koh and A. I. Bush, Nat. Rev. Neurosci.,
2005, 6, 449.
4 L. A. Finney and T. V. O’Halloran, Science, 2003, 300, 931.
5 A. Krezel and W. Maret, JBIC, J. Biol. Inorg. Chem., 2006, 11, 1049.
6 W. Maret, BioMetals, 2001, 14, 187.
7 D. H. Nies, Science, 2007, 317, 1695.
8 A. Krezel, Q. Hao and W. Maret, Arch. Biochem. Biophys., 2007,
463, 188.
Fig. 4 Green channel: 1 (top), [Zn(1)]2+ (bottom). Red channel:
(A) Fusion of mApple fluorescent protein with an endosomal targeting
peptide. (B) mApple fused to a Golgi targeting peptide. (C) mApple
fused to lysosomal membrane glycoprotein 1 (LAMP1). (D) MitoTracker
Red CMXRos. Scale bar: 10 mm.
9 K. Kikuchi, H. Komatsu and T. Nagano, Curr. Opin. Chem. Biol.,
2004, 8, 182.
10 R. B. Thompson, Curr. Opin. Chem. Biol., 2005, 9, 526.
11 C. J. Chang and S. J. Lippard, Met. Ions Life Sci., 2006, 1, 61.
12 Z. Dai and J. W. Canary, New J. Chem., 2007, 31, 1708.
13 E. M. Nolan and S. J. Lippard, Acc. Chem. Res., 2009, 42, 193.
14 S. Y. Assaf and S.-H. Chung, Nature, 1984, 308, 734.
15 K. Komatsu, K. Kikuchi, H. Kojima, Y. Urano and T. Nagano,
J. Am. Chem. Soc., 2005, 127, 10197.
16 E. M. Nolan, J. Jaworski, K.-I. Okamoto, Y. Hayashi, M. Sheng
and S. J. Lippard, J. Am. Chem. Soc., 2005, 127, 16812.
17 M. D. Shults, D. A. Pearce and B. Imperiali, J. Am. Chem. Soc.,
2003, 125, 10591.
18 C. R. Goldsmith and S. J. Lippard, Inorg. Chem., 2006, 45, 555.
19 P. Paoletti, A. M. Vergnano, B. Barbour and M. Casado,
Neuroscience, 2009, 158, 126.
20 L. Zhang, S. Dong and L. Zhu, Chem. Commun., 2007, 1891.
21 S. Huang, R. J. Clark and L. Zhu, Org. Lett., 2007, 9, 4999.
22 L. Zhang, R. J. Clark and L. Zhu, Chem.–Eur. J., 2008, 14, 2894.
23 L. Zhang, W. A. Whitfield and L. Zhu, Chem. Commun., 2008, 1880.
24 L. Zhang and L. Zhu, J. Org. Chem., 2008, 73, 8321.
25 L. Zhu, L. Zhang and A. H. Younes, Supramol. Chem., 2009, 21,
268.
26 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.
27 J. Cody, S. Mandal, L. Yang and C. J. Fahrni, J. Am. Chem. Soc.,
2008, 130, 13023.
28 R. Parkesh, T. C. Lee and T. Gunnlaugsson, Org. Biomol. Chem.,
2007, 5, 310.
29 B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3.
30 E. Kawabata, K. Kikuchi, Y. Urano, H. Kojima, A. Odani and
T. Nagano, J. Am. Chem. Soc., 2005, 127, 818.
The physiological significance of mitochondrial zinc has been
investigated using zinc-selective fluorescent probes.35 Evidence
suggests that mitochondria function as independently operated
endogenous zinc pools which are both sources and sinks
during zinc homeostasis.35 The availability of mitochondria-
targeting zinc probes with broad concentration coverages is
expected to facilitate the investigations of zinc transport
between several intracellular zinc pools (e.g. mitochondrial,
vesicular, and ligand/protein bound).
The localization efficiencies of 1 in other organelles were
studied via co-staining experiments using organelle-specific
fusions to the red fluorescent protein, mApple.36 Compound
1 extensively localizes in Golgi complexes and lysosomes as
indicated by Pearson’s coefficients over 0.50. 1 does not
accumulate in endosomes efficiently (PC = 0.33), suggesting
that it might have entered the cell via passive permeation
through the cell membrane rather than endocytosis. With
addition of 100 mM ZnCl2, the localization properties of 1
vary. In particular, a very high Pearson’s coefficient for
lysosomes (0.93) was recorded. The increase of localization
of 1 in lysosomes at cytotoxic zinc levels suggests that cells
have transported surplus zinc ions to lysosomes for disposal.
In summary, we have demonstrated that through rational
design fluorescent heteroditopic ligands for zinc ions can be
prepared whose dual-channel fluorescence is capable of correlating
[Zn]f under simulated physiological conditions over a range of
6 orders of magnitude. The preliminary live-cell imaging studies
showed that compound 1 (a) is able to penetrate the cell
membrane readily, (b) is not overly toxic to HeLa cells during
the course of the experiment, (c) accumulates in mitochondria
and to lesser extents in other organelles, and (d) undergoes
fluorescence enhancement with increasing zinc gradient. Based
on our understanding of the coordination-driven photophysical
processes in this heteroditopic platform, we are currently
preparing variants of compound 1 with lower excitation energies
to reduce the likelihood of autofluorescence from cellular
samples, and larger spectral separations of two wavelength
channels so that the intensity at each channel can be collected
using matching filter sets in live-cell imaging experiments.
31 Ca2+ and Mg2+ show little effect on the fluorescence of 1, which
ensures the utility of 1 in intracellular imaging. In general, first-row
transition metal ions have high affinities to polyaza ligands such as
1, most of which (e.g. Cu2+) quenches fluorescence. Cd2+ which
belongs to the same group with Zn2+ elicits similar fluorescence
response from 1 (Fig. S8w). Despite the coordination promiscuity
of polyaza ligands, they are frequently incorporated in intracellular
fluorescent indicators because of the trace nature of the interfering
ions in a cellular environment.
32 R. A. Bozym, R. B. Thompson, A. K. Stoddard and C. A. Fierke,
ACS Chem. Biol., 2006, 1, 103.
33 A. Smallcombe, BioTechniques, 2001, 30, 1240.
34 F. Pierrel, P. A. Cobine and D. R. Winge, BioMetals, 2007, 20, 675.
35 S. 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.
36 N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach,
K. L. Hazelwood, M. W. Davidson and R. Y. Tsien, Nat. Methods,
2008, 5, 545.
ꢀc
This journal is The Royal Society of Chemistry 2009
7410 | Chem. Commun., 2009, 7408–7410