A selective fluorescent sensor for detecting lead in living cells

Article abstract of DOI:10.1021/ja063029x

We present the synthesis, properties, and biological applications of Leadfluor-1 (LF1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively imaging Pb²⁺ in aqueous solution and in living cells. LF1 combines a fluoresc

Full text of DOI:10.1021/ja063029x

Published on Web 06/29/2006
A Selective Fluorescent Sensor for Detecting Lead in Living Cells
Qiwen He, Evan W. Miller, Audrey P. Wong, and Christopher J. Chang*
Department of Chemistry, UniVersity of California, Berkeley, California 94720
Received May 1, 2006; E-mail: chrischang@berkeley.edu
Scheme 1. Synthesis of Leadfluor-1 (LF1)
Lead pollution is an ongoing danger to human health and the
environment, as most of the 300 million tons of this heavy metal
mined to date are still circulating in soil and groundwater.1 Despite
efforts to reduce global emissions, lead poisoning remains the
world’s most common environmentally caused disease.2 Once
introduced into the body, lead is a potent neurotoxin that can
interfere with brain development,3 slow nerve conduction velocity,4
and trigger behavioral problems.5 Plausible molecular targets of
lead include calcium- and zinc-binding proteins that control cell
signaling and gene expression, respectively,2,6-10 but mechanisms
of lead toxicity remain an open question for study.
Interest in elucidating these pathways, as well as public concerns
over toxic lead exposure, provides a need for devising new ways
to track Pb2+ in natural samples. Whereas standard techniques, such
as atomic absorption or anodic stripping voltammetry, can only
measure total lead content, fluorescence imaging with Pb2+-sensitive
chemosensors can, in principle, provide information on bioavailable
lead pools with spatial and temporal resolution. The major
challenges to achieving this goal are creating systems that are
selective for Pb2+ and can function in water. Pb2+-responsive
fluorescent probes based on peptide,11 protein,12 DNAzyme,13-16
polymer,17 and small-molecule18-27 scaffolds have been reported.
However, none of these systems have been utilized successfully
for tracking Pb2+ in living cells, as current probes can be limited
by interfering background fluorescence or nonspecific quenching
from competing metal ions, incompatibility with water, and/or
ultraviolet excitation that can damage or trigger autofluorescence
from biological samples. We now present the synthesis and
properties of Leadfluor-1 (LF1, 6), a new turn-on fluorescent sensor
for selective detection of Pb2+ in water and in living cells. LF1
features visible wavelength excitation and emission profiles and a
ca. 18-fold fluorescence enhancement upon Pb2+ binding. Moreover,
confocal microscopy experiments establish that LF1 can monitor
changes in Pb2+ levels within living mammalian cells.
LF1 combines a fluorescein-type scaffold with attractive optical
properties and biological compatibility with a dicarboxylate
pseudocrown receptor designed to satisfy the size and charge
requirements of the Pb2+ cation.28 The synthetic route to LF1 is
shown in Scheme 1. Reaction of 2-anilinoethanol with 2-(2-
chloroethoxy)ethanol affords diol 1 in 66% yield. Alkylation of 1
with bromoacetic acid followed by acid-catalyzed esterification
provides diester 2 in 61% yield. Vilsmeier formylation of 2 using
POCl3/DMF followed by basic workup generates aldehyde 3 in 50%
yield, which is then coupled with 2 equiv of resorcinol to produce
the tetrahydroxy intermediate 4 in 14% yield. DDQ oxidation of 4
furnishes xanthenone 5 in 41% yield. Ester deprotection of 5 under
basic conditions proceeds quantitatively to give LF1.
of Pb2+, the fluorescence intensity of LF1 increases by ca. 18-fold
(Φ ) 0.013, Figure 1a) with the same absorption (λabs ) 490 nm,
ꢀ ) 2.8 × 104 M-1 cm-1) and emission maxima (λem ) 514 nm)
as the apo probe. The turn-on response is reversible; treatment with
the chelator TPEN restores LF1 fluorescence back to within 5% of
baseline levels. Binding assays using the method of continuous
variations (Job’s plot) are consistent with a 1:1 Pb2+:LF1 complex
being responsible for the observed fluorescence enhancement, and
the Kd for Pb2+ coordination to LF1 is 23 ( 4 µM. LF1 is capable
of detecting environmentally relevant concentrations of aqueous
Pb2+. The addition of 15 ppb Pb2+, the maximum EPA limit for
allowable level of lead in drinking water, to a 5 µM solution of
LF1 triggers a 15 ( 2% increase in fluorescence intensity.
LF1 also exhibits a selective turn-on fluorescence response to
Pb2+ in aqueous solution. Responses of 5 µM LF1 to the presence
of various biologically and environmentally relevant metal ions are
collected in Figure 1b. The fluorescence profiles of apo or Pb2+
-
bound LF1 are unchanged in the presence of 2 mM Li+, Na+, K+,
Mg2+, and Ca2+, indicating excellent selectivities for Pb2+ over these
alkali and alkaline earth cations. A series of 3d metal ions, including
75 µM Mn2+, Fe2+, Co2+, Ni2+, and Zn2+, do not trigger LF1
fluorescence enhancements or interfere with its Pb2+ response. Of
the first-row transition metal ions, only Cu2+ at 75 µM can limit
the turn-on Pb2+ response of LF1, but lower concentrations of Cu2+
(25 µM) do not interfere. LF1 also shows selectivity for Pb2+ over
the heavy metal ions Hg2+ and Cd2+
.
Subsequent experiments probed the ability of LF1 to track Pb2+
levels in living cells using confocal microscopy. HEK cells
incubated with 20 µM of LF1-AM for up to 90 min at 37 °C show
negligible intracellular fluorescence (Figure 2a). In contrast, LF1-
stained cells exposed to 200 µM Pb2+ for 40 min at 37 °C display
enhanced cytosolic fluorescence (Figure 2b). Treatment of cells
Spectroscopic measurements with LF1 were performed under
simulated physiological conditions (20 mM HEPES, buffer pH 7).
LF1 displays a characteristic fluorescein-like absorption band in
the visible region centered at 490 nm (ꢀ ) 2.5 × 104 M-1 cm-1
)
and weak fluorescence (Φ < 0.001, λem ) 514 nm). Upon addition
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10.1021/ja063029x CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
that LF1 can respond to changes in intracellular Pb2+ levels within
living mammalian cells.
In conclusion, LF1 is a new type of synthetic fluorescent sensor
for probing Pb2+ in living biological samples. Desirable features
of this fluorescein-based reagent include a selective turn-on response
for Pb2+ over competing metal ions in water, sensitivity to EPA
limits of lead poisoning, visible excitation and emission profiles to
minimize cellular autofluorescence and photodamage, and the ability
to track changes in Pb2+ levels within living cells. Immediate goals
to expand the utility of LF1 and related chemosensor platforms for
studies of lead biology include optimization of sensitivities and
selectivities to Pb2+, as well as improvement of optical brightness
values and dynamic ranges for imaging applications.
Acknowledgment. We thank the University of California,
Berkeley, the Camille and Henry Dreyfus Foundation, the Arnold
and Mabel Beckman Foundation, the American Federation for
Aging Research, and the NSF (CAREER Award CHE-0548245)
for funding this work. E.W.M. was supported by a Chemical
Biology Interface Training Grant from the NIH (T32 GM066698).
We thank Ms. Sarah Bell for preliminary experiments.
Supporting Information Available: Synthetic and experimental
details (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
Figure 1. (a) Fluorescence response of 5 µM LF1 to Pb2+. Spectra shown
are for Pb2+ concentrations of 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 35, 50, and
75 µM. Spectra were acquired in 20 mM HEPES, pH 7, with excitation at
490 nm. (b) Fluorescence responses of 5 µM LF1 to various metal ions.
Bars represent the final integrated fluorescence response (Ff) over the initial
integrated emission (Fi). Spectra were acquired in 20 mM HEPES, pH 7.
White bars represent the addition of an excess of the appropriate metal ion
(2 mM for Li+, Na+, K+, Mg2+, and Ca2+, 75 µM for all other cations
except entry 11) to a 5 µM solution of LF1. Black bars represent the addition
of 75 µM Pb2+ to the solution. Excitation was provided at 490 nm, and the
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