A turn-on fluorescent sensor for detecting nickel in living cells

Article abstract of DOI:10.1021/ja906500m

(Figure Presented) We present the synthesis and properties of Nickelsensor-1 (NS1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively responding to Ni²⁺ in aqueous solution and in living cells. NS1 combines a BODIPY

Full text of DOI:10.1021/ja906500m

Published on Web 12/01/2009
A Turn-On Fluorescent Sensor for Detecting Nickel in Living Cells
Sheel C. Dodani, Qiwen He, and Christopher J. Chang*
Department of Chemistry and the Howard Hughes Medical Institute, UniVersity of California,
Berkeley, California 94720
Received August 1, 2009; E-mail: chrischang@berkeley.edu
Scheme 1. Synthesis of Nickelsensor-1 (NS1)
Nickel is an essential metal nutrient for supporting life, but loss
of nickel homeostasis is harmful to prokaryotic and eukaryotic
organisms alike.1 Elegant studies continue to elucidate mechanisms
for Ni2+ uptake, regulation, and efflux,2-10 as well as to define the
redox and nonredox roles of nickel biochemistry in microbial and
plant systems.11-17 However, the contributions of nickel homeo-
stasis to mammalian health and disease remain largely unexplored.18
In this context, excess nickel accumulation can aberrantly affect
respiratory and immune systems, but mechanisms of nickel imbal-
ance are insufficiently understood.19,20
To help elucidate the roles of nickel in living systems, we are
developing Ni2+-selective fluorescent indicators as part of a larger
program aimed at studying metals in biology by molecular
imaging.21,22 Such chemical tools, in principle, can be used to
monitor exchangeable nickel pools with spatial and temporal
resolution and provide a complement to standard bulk techniques
for measuring total nickel content such as atomic absorption or
inductively coupled plasma mass spectrometry. A major chemical
challenge to this end is designing systems with Ni2+-specific
responses over other biologically relevant metal ions in water.
507 nm) compared to the apo probe. The turn-on response is
reversible; treatment of Ni2+-loaded NS1 with the divalent metal
ion chelator TPEN restores NS1 fluorescence back to baseline
levels. A Hill plot indicates a simple binding process with no
cooperativity (Figure S1a), and the apparent Kd for Ni2+ binding
to NS1 is 193 ( 5 µM (Figure S1b).
Examples of Ni2+-responsive fluorescent probes remain rare; Ni2+
-
NS1 exhibits a selective turn-on fluorescence response to Ni2+
in water. Responses of 2 µM NS1 to the presence of various
biologically relevant metal ions are shown in Figure 1b. The
fluorescence profiles of apo or Ni2+-bound NS1 are unchanged in
the presence of 1 mM Na+, K+, Mg2+, and Ca2+, indicating
excellent selectivities for Ni2+ over these alkali and alkaline earth
cations. Moreover, a series of 3d divalent metal cations, including
100 µM Mn2+, Fe2+, Co2+, and Zn2+, do not trigger NS1
fluorescence enhancements or interfere with the Ni2+ response. Of
the first-row divalent transition metal ions, Cu2+ at 100 µM can
mute the turn-on Ni2+ response of NS1, but lower Cu2+ levels (2
µM) minimize this interference. As expected, Cu2+ binds the sensor
due to Irving-Williams series considerations, but the paramagnetic
d9 ion quenches fluorescence, suggesting that the fluorescence
increase observed for Ni2+ is due to a diamagnetic d8 state.
We next established the ability of NS1 to track Ni2+ levels in
living cells using a model for respiratory nickel exposure. Live-
cell confocal microscopy imaging experiments utilized the ac-
etoxymethyl ester form of NS1 (NS1-AM) to enhance membrane
permeability. Live human lung carcinoma A549 cells loaded with
a 1:1 (v/v) mixture of NS1-AM and Pluronic F-127 (10 µM) for
35 min at 37 °C show weak intracellular fluorescence (Figure 2a).
A549 cells supplemented with 1 mM NiCl2 in the growth medium
for 18 h at 37 °C and then stained with NS1 under the same loading
conditions results in an increase in observed intracellular fluores-
cence intensity (Figure 2b); previous experiments establish that this
exposure level of nickel is not lethal to lung carcinoma cells,
whereas 2-10 mM Ni2+ causes widespread cell death.35 Treatment
of cells loaded with NS1 and Ni2+ with the divalent metal chelator
TPEN (1 mM) for 1 min at 25 °C reverses the observed fluorescence
increases (Figure 2c). Finally, Hoescht-3342 staining confirms that
selective peptide,23,24 protein,25 polymer,26,27 and small-molecule
based sensors28-30 have been reported but have not been utilized
for cellular imaging, whereas the commercial Zn2+ sensor Newport
Green DCF also responds to Ni2+ and Ti3+ and has been used to
detect their accumulation in cells.31-34 In this report, we present
the synthesis and properties of Nickelsensor-1 (NS1, 5), a new turn-
on fluorescent sensor for the selective detection of Ni2+ in water
and in biological samples. NS1 features visible wavelength spectral
profiles and a ca. 25-fold fluorescence increase upon Ni2+ binding.
Confocal microscopy experiments show that this indicator can
reliably monitor changes in Ni2+ levels within living mammalian
cells.
Our design for NS1 combines a BODIPY dye reporter with a
mixed N/O/S receptor to satisfy the Ni2+ cation (Scheme 1).
Addition of ditosylate 1 to Cs2CO3 and methyl thioglycolate affords
diester 2 in 41% yield. Vilsmeier formylation of 2 using POCl3/
DMF followed by basic workup furnishes aldehyde 3 in 60% yield.
BODIPY 4 is obtained in a one-pot, three-step procedure via
condensation of 3 with 2,4-dimethylpyrrole, followed by DDQ
oxidation and boron insertion with BF3•OEt2 (38% overall yield
for three steps). Ester hydrolysis of 4 under basic conditions gives
NS1 (5) in 71% yield.
Spectroscopic evaluation of NS1 was performed in 20 mM
HEPES buffered to pH 7.1. The optical features of the probe are
characteristic of the BODIPY platform. Apo NS1 displays one
visible region absorption band centered at 495 nm (ꢀ ) 5.8 × 103
M-1 cm-1) and an emission maximum at 507 nm (Φ ) 0.002).
Addition of 50 equiv of Ni2+ triggers a ca. 25-fold fluorescence
turn-on (Φ ) 0.055, Figure 1a) with no shifts in absorption (λabs
) 495 nm, ꢀ ) 5.5 × 103 M-1 cm-1) or emission maxima (λem
)
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10.1021/ja906500m  2009 American Chemical Society

C O M M U N I C A T I O N S
metal ions. Confocal microscopy experiments show that NS1 can
be used for detecting changes in Ni2+ levels within living cells.
Future plans will focus on improving the optical brightness and
binding affinities of this first-generation probe as well as applying
NS1 and related chemical tools to probe the cell biology of nickel.
Acknowledgment. We thank the Dreyfus, Packard, and Sloan
Foundations, the Hellman Faculty Fund, Amgen, NSF (CAREER
CHE-0548245), NIH (GM 79465), and HHMI for providing funding
for this work. We thank Holly Aaron (UCB Molecular Imaging
Center) and Ann Fischer and Michelle Yasukawa (UCB Tissue
Culture Facility) for expert technical assistance.
Supporting Information Available: Synthetic and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 1. (a) Fluorescence response of 2 µM NS1 to Ni2+. Spectra shown
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