A highly selective turn-on colorimetric, red fluorescent sensor for detecting mobile zinc in living cells

Article abstract of DOI:10.1021/ic101569a

We describe ZRL1, a turn-on colorimetric and red fluorescent zinc ion sensor. The Zn²⁺-promoted ring opening of the rhodamine spirolactam ring in ZRL1 evokes a 220-fold fluorescence turn-on response. In aqueous media, ZRL1 turn-on luminescence is highly selective for Zn²⁺ ions, with no significant response to other competitive cations, including Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe ²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd ²⁺, or Hg²⁺. In addition to these characteristics, preliminary results indicate that ZRL1 can be delivered to living cells and can be used to monitor changes in intracellular Zn²⁺ levels.

Full text of DOI:10.1021/ic101569a

Inorg. Chem. 2010, 49, 10753–10755 10753
DOI: 10.1021/ic101569a
A Highly Selective Turn-On Colorimetric, Red Fluorescent Sensor for Detecting
Mobile Zinc in Living Cells
Pingwu Du and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue,
Cambridge, Massachusetts 02139, United States
Received August 3, 2010
We describe ZRL1, a turn-on colorimetric and red fluorescent zinc
ion sensor. The Zn2þ-promoted ring opening of the rhodamine
spirolactam ring in ZRL1 evokes a 220-fold fluorescence turn-on
response. In aqueous media, ZRL1 turn-on luminescence is highly
selective for Zn2þ ions, with no significant response to other
Cu2þ, Hg2þ, Pb2þ, Au3þ, and Agþ.10-20 The spirocyclic
forms of these dyes, which are colorless and nonfluorescent,
become colorful and strongly fluorescent when cations react
with the sensors. To our knowledge, there are only two
examples of zinc sensing based on such a rhodamine dye
spirocyclic ring-opening mechanism.21,22 One, termed Rhoda-P,
is selective forzinc, but its properties were investigatedonly in
organic solvents and there was no information about its
reversibility or applicability for live cell imaging.21 Moreover,
we find that Rhoda-P evokes no appreciable turn-on re-
sponse to zinc ions in buffered aqueous solution (Figure S1,
Supporting Information, SI). The other example is a ratio-
metric zinc sensor based on a hybrid fluorescein-rhodamine
lactam platform.22 Its photophysical properties were investi-
gated in 50% aqueous acetonitrile solution, and there was
fluorescence interference by [Hg2þ] > 20 μM and protons in
the pH range 6.0-7.0.
competitive cations, including Naþ, Kþ, Ca2þ, Mg2þ, Mn2þ
,
Fe2þ, Co2þ, Ni2þ, Cu2þ, Cd2þ, or Hg2þ. In addition to these
characteristics, preliminary results indicate that ZRL1 can be
delivered to living cells and can be used to monitor changes in
intracellular Zn2þ levels.
Mobile zinc plays an important role in life processes, and
disruption of zinc homeostasis has been implicated in health
disorders including Alzheimer’s disease1,2 and diabetes.3
Methods for sensing Zn2þ uptake, accumulation, trafficking,
and efflux have therefore attracted much recent attention.4,5
Although several Zn2þ-selective fluorescent sensors are now
available,6-9 novel constructs remain in high demand to
provide one or more of the following features: (1) selectivity
for Zn2þ; (2) pH-independent response under physiological
conditions; (3) large dynamic range; (4) red or near-infrared
emission to avoid autofluorescence by biomolecules; (5)
improved photostability; and (6) ratiometric detection.
Rhodamine B and its derivatives, because of their high
quantum yields and good photostability, are of particular
interest for cation sensing. The mechanism of fluorescent
turn-on for these compounds typically involves opening of a
In the present Communication, we describe the synthesis
and properties of a water-soluble, turn-on red fluorescent
sensor, ZinRhodaLactam-1 (ZRL1), which exhibits ex-
tremely low background fluorescence and a large turn-on
response when bound to zinc in aqueous media. ZRL1 has
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Zhou, Z.; Yi, T.; Huang, C. Chem. Eur. J. 2008, 14, 6892–6900.
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spirolactam ring by external reagents, including Cr3þ, Fe3þ
,
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12, 401–403.
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W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, 10107–10111.
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*To whom correspondence should be addressed. E-mail: lippard@
mit.edu.
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r
2010 American Chemical Society
Published on Web 10/28/2010
pubs.acs.org/IC

10754 Inorganic Chemistry, Vol. 49, No. 23, 2010
Scheme 1. Synthesis of ZRL1, ZRL2, and ZRL3
Du and Lippard
Figure 1. ORTEP diagram of ZRL1 showing 50% probability thermal
ellipsoids on all non-hydrogen atoms. Hydrogen atoms are omitted for
clarity. Oxygen atoms are red, carbons are white, and nitrogens are blue.
excellent selectivity, is pH-independent in the range 5.4-11.0,
and exhibits a large dynamic range for mobile Zn2þ. Fluor-
escence microscopy experiments further establish that ZRL1
is membrane-permeable and can be used to monitor intra-
cellular Zn2þ in live cells.
The design of ZRL1 combines a rhodamine B nonfluor-
escent lactam and a zinc-binding ligand, 2-[bis(2-pyridyl-
methyl)aminomethyl]aniline (1; Scheme 1). ZRL1 was syn-
thesized by treating rhodamine B base with POCl3, followed
by dropwise addition to a solution of 1, prepared by our pub-
lished method.23 After column chromatography (alumina,
eluent 50:1 (v/v) CH2Cl2/MeOH), ZRL1 was obtained in
17% yield and was quite stable over 6 months, even when
kept at room temperature. The analogues ZRL2 and ZRL3
were prepared by the same route, except that 3-[bis(2-pyr-
idylmethyl)aminomethyl]aniline (2) and 4-[bis(2-pyridyl-
methyl)aminomethyl]aniline (3) were used in place of 1.
The synthetic procedures for compounds 2 and 3 are similar
to those for 1, as fully described in the SI. ZRL1, ZRL2, and
ZRL3 were characterized by 1H and 13C NMR spectroscopic
and HRMS methods, and the structure of ZRL1 was deter-
mined by X-ray crystallography (Figure 1 and Tables S1
and S2, SI).
ZRL1 forms colorless and nonfluorescent solutions in
either aqueous media or organic solvents, including DMSO,
acetone, MeOH, or CH3CN. Addition of zinc ions to ZRL1
in aqueous solution or organic solvents leads to the develop-
ment of a pink color and red fluorescence, indicating that
ZRL1 can detect zinc ions via opening of the spirolactam
ring. Quantitative photophysical studies were conducted
under simulated physiological conditions (50 mM PIPES
with 100 mM KCl, pH 7.0). The absorption and fluorescence
spectral titrations by zinc ions are shown in Figure 2. The
metal-free sensor initially displayed no appreciable absorp-
tion or emission band above 500 nm (Φ < 0.001), indicating
the existence of only the lactam form in aqueous media.
Addition of Zn2þ ions triggered formation of a visible
absorption band centered at 569 nm and an emission band
with a maximum at 595 nm. The solution changed from
colorless to pink. These results indicate that Zn2þ ions
promote the transformation of ZRL1 from the lactam to
Figure 2. Absorption (left) and fluorescence (right) spectra of ZRL1
(10 μM) in a buffered solution upon addition of different amounts of
ZnCl2. Excitation was performed at 550 nm. The inset pictures show,
following addition of100 equiv ofzincions toZRL1(10 μM) in a buffered
solution, visible color formation by the naked eye (left) and fluorescence
(right) under illumination by a hand-held UV lamp. All spectra were
acquired 2 h after mixing ZRL1 with zinc in 50 mM PIPES at pH 7.0
containing 100 mM KCl.
ring-opened form. Incremental addition of Zn2þ [(0-1.8) ꢀ
10-3 M] provided a steady fluorescence turn-on with no shift
in either the absorption or emission maximum. When the
concentration of Zn2þ was greater than 1.0 ꢀ 10-3 M, the
optical features reached a plateau, with ε569 ∼ 20800 M-1
cm-1 and the emission quantum yield ΦFL = 0.22. The
∼220-fold increase in the fluorescence quantum yield pro-
vides an excellent turn-on ratio for zinc sensing in aqueous
media. Moreover, the response of ZRL1 to zinc ions can be
reversed. Titration of the ZRL1-Zn2þ complex with a metal
ion chelator, either TPEN or EDTA, restored the fluores-
cence to baseline levels (Figures S2 and S3,SI). The effect of
pH on the fluorescence spectrum of ZRL1 was also investi-
gated in aqueous media (Figure S4, SI). In the pH range from
5.4 to 11.0, the zinc-induced ZRL1 fluorescence is pH-
independent and the free ligand has no appreciable emission
in the visible region of the spectrum. This property is valuable
for applications to living cells, where pH changes are caused
by biological stimuli.
For comparison, the photophysical properties of ZRL2
and ZRL3 were also investigated in buffer (50 mM PIPES
containing 100 mM KCl, pH 7.0). However, both com-
pounds failed to exhibit any appreciable turn-on response
to Zn2þ ions (Figures S5 and S6, SI), indicating the impor-
tance of close proximity between the carbonyl group on the
lactam ring and the adjacent dipicolylamine zinc-chelating
moiety for ring opening of the lactam.
The ZRL1 zinc binding affinity was analyzed by fluores-
cence spectroscopy. The fit to a plot of normalized intensity
(23) Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am.
Chem. Soc. 2003, 125, 1778–1787.
versus [Zn2þ
]total (Figure S7, SI) revealed a turn-on constant

Communication
Inorganic Chemistry, Vol. 49, No. 23, 2010 10755
Figure 4. Live-cell imaging of HeLa cells after incubation with ZRL1
(10 μM) and Hoechst 33258 nuclear stain (blue): (a) Bright-field transmis-
sion image. (b) Nuclear image stained by Hoechst 33258. (c) Fluorescence
image without addition of Zn2þ. (d) Fluorescence image of cells incubated
with ZRL1 for 15 min, washed twice, and then treated with 50 μM Zn2þ
pyrithione for 20 min. (e) ZRL1-loaded, 50 μM Zn2þ-supplemented cells
treated with 80 μM TPEN after 10 min. Scale bar = 25 μm.
Figure 3. Fluorescence spectroscopic response of ZRL1 to various
metal ions. All spectra were acquired after 2 h upon mixing ZRL1 with
zinc in 50 mM PIPES at pH 7.0 containing 100 mM KCl. Normalized
fluorescence intensities represent the addition of an excess of the appro-
priate metal ion (1 mM for Naþ, Kþ, Ca2þ, and Mg2þ; all others are
200 μM) toa10μMsolutionofZRL1. Excitationwasprovided at550nm.
Kt of 73 ( 2 μM, where Kt represents both zinc dissociation
and lactam formation, for ZRL1 binding to Zn2þ. The Kt
value of ZRL1 is at the high end compared to Kd values of
many related sensors with dipicolylamine Zn2þ-binding
groups8 but in the functional range for sensing of the ion in
many neurochemical applications.2,5
ZRL1 for 15 min at 37 °C exhibit a weak intracellular red
fluorescence (Figure 4c). After addition of 50 μM Zn2þ
carried into the live cells by the ionophore pyrithione, the
red fluorescence was much stronger and clearly observable
(Figure 4d). The red fluorescence substantially decreased
upon introduction of 80 μM TPEN (Figure 4e). Figure 4b
shows cells treated with the nuclear stain Hoescht 33258.
These results indicate that ZRL1 is cell-permeable and can be
used to image Zn2þ within living cells.
In conclusion, ZRL1 based on rhodamine B in its lactam
form undergoes a large, turn-on red fluorescence specifically
in the presence of zinc ions in aqueous media and in live cells.
The increase in quantum yield on binding zinc is ∼220-fold,
and the fluorescence can be reversed upon addition of an
appropriate zinc chelator. These properties will make ZRL1
of value in studies of the neurochemical functions of mobile
zinc that are currently in progress.
The fluorescence turn-on of ZRL1 is highly selective for
Zn2þ over many other monovalent and divalent metal ions
(Figures 3 and S8, SI). Even large excesses of Naþ, Kþ, Ca2þ
,
and Mg2þ (1 mM), the most biologically relevant, potentially
competing metal ions, barely perturb the fluorescence of the
zinc-free and zinc-bound forms of ZRL1. Moreover, addi-
tion of 200 μM concentrations of any of the following
divalent transition-metal ions into ZRL1, Mn2þ, Fe2þ
,
Co2þ, Ni2þ, Cu2þ, Cd2þ, or Hg2þ, does not induce an
appreciable fluorescence turn-on, although Co2þ, Ni2þ, and
Cu2þ show an obvious absorption response (Figure S9, SI).
The paramagnetism of these last three ions most likely
quenches the fluorescence of the resulting ZRL1 complexes.
Thus, only Zn2þ evokes a fluorescent turn-on response over all
other competing metal ions tested. Many reported fluorescent
Zn2þ sensors, such as those in the Zinpyr family,8 lanthanide
chemosensors,24 and some ZnAF family members,25 cannot
distinguish Zn2þ from Cd2þ and/or Hg2þ. At present, we do
not have a good explanation for this behavior.
Acknowledgment. This work was supported by a grant
from the National Institute of General Medical Sciences
(GM065519 to S.J.L.). We thank Dr. E. Tomat for
assistance with microscopy, Dr. Y. You for help with
the determination of Kt, and Drs. E. Tomat and D.
Buccella for helpful comments on the manuscript.
The ability of ZRL1 to track zinc ions in living cells was
also examined (Figure 4). HeLa cells incubated with 10 μM
Supporting Information Available: Full synthetic procedures,
characterization data, crystallographic details in CIF format of
ZRL1, pH titration of ZRL1, and data fitting method for
fluorescence turn-on constant. This material is available free
of charge via the Internet at http://pubs.acs.org.
(24) Hanaoka, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T.
J. Am. Chem. Soc. 2004, 126, 12470–12476.
(25) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T.
J. Am. Chem. Soc. 2005, 127, 10197–10204.