5644
J. Am. Chem. Soc. 2000, 122, 5644-5645
protein-based19 scaffolds, as well as fluorophore-appended
macrocycles.20-22 Although each has potential advantages over
the quinoline probes, none has been successfully applied to
measure Zn2+ within living cells, either because of unacceptable
binding affinity, optical properties, or the need to perform
microinjections for their use. Probes traditionally used to measure
Ca2+ or Mg2+ have also been employed, but the signals from
A New Cell-Permeable Fluorescent Probe for Zn2+
Grant K. Walkup,† Shawn C. Burdette,‡
Stephen J. Lippard,*,‡ and Roger Y. Tsien*,†,§
Department of Pharmacology and
Chemistry and Biochemistry and
Howard Hughes Medical Institute
UniVersity of California at San Diego
La Jolla, California 92093-0647
Department of Chemistry
these species are hard to separate from those of Zn2+ 23,24
.
To achieve high affinity binding without quinoline sulfonamide
or EGTA-based chelating moieties, we incorporated the bis(2-
pyridylmethyl)amine (di-2-picolylamine or DPA) moiety into our
probe to chelate zinc. The [Zn(DPA)]2+ complex has an apparent
Kd of 70 nM at pH 7 and essentially no measurable affinity for
Massachusetts Institute of Technology
Cambridge, Massachusetts, 02139
ReceiVed March 10, 2000
ReVised Manuscript ReceiVed April 25, 2000
Ca2+ or Mg2+ 25
. Furthermore, the DPA ligand is not expected to
present a challenge to membrane permeability, owing to its struc-
tural similarity to the membrane-permeant heavy metal chelator
N,N,N′,N′-tetra(2-picolyl)ethylenediamine (TPEN).26 Finally we
selected fluorescein as the reporting group because of its large
extinction coefficient, high quantum yield, membrane per-
meability, and the ready availability of optical filter sets for
fluorescence microscopy.
Access to a DPA-derivatized fluorescein was achieved through
a Mannich reaction between 2′,7′-dichlorofluorescein (DCF) and
the iminium ion condensation of product of formaldehyde and
DPA. The chlorines in DCF restrict substitution to the 4′- and
Zinc is an essential cofactor, critical for numerous cellular func-
tions.1 The combination of its unique chemical properties2 and
its central role in processes including gene expression,3 apoptosis,4
enzyme regulation,5 and neurotransmission6,7 suggests that Zn2+
may be a major regulatory ion in the metabolism of cells.8,9
Although Zn2+ is abundant in eukaryotes and most is tightly
bound, pools of chelatable Zn2+ have been imaged in living cells
with concentrations ranging from sub-nM in undifferentiated
mammalian cells10 to ∼0.3 mM in hippocampal nerve synaptic
vesicles.11 Currently, the most widely applied probes for cellular
zinc are aryl sulfonamides of 8-aminoquinoline such as 6-meth-
oxy-(8-p-toluenesulfonamido)quinoline (TSQ),12 Zinquin4,13 and
TFLZn11 (TSQ analogues). The distinction between chelatable
and free Zn2+ is problematic due to the fact that these quinoline-
based dyes can form mixed complexes, sensing Zn2+ that is
already partially coordinated. Recent studies have clarified some
of the details regarding the aqueous Zn2+-binding equilibria of
such dyes, enhancing their value as quantitative probes.14 Quino-
line probes require ultraviolet excitation (∼350 nm), however,
which can be damaging to cells, and have relatively dim
fluorescence with quantum yields ≈ 0.1 and extinction coefficients
≈ 10 × 103 M-1 cm-1 15
.
5′-positions of the fluorescein ring, lower the phenolic pKa so
that it is largely deprotonated at physiological pH, and provide a
small redshift to the chromophore. The resulting molecule, termed
Zinpyr-1, displays a highly Zn2+-selective fluorescence response.27
Because the availability of better Zn2+-specific probes would
provide additional insight into the cell biology of Zn2+, interest
in the field remains high. Several fluorescent sensors for Zn2+
have been described, including those with polypeptide16-18 or
1
Analysis of the crude material (TLC, H NMR) suggests that it
is obtained in >80% purity after trituration from boiling ethanol.
Material of analytical purity used for all of the present studies is
obtained in 46% yield after chromatography on alumina or reverse
phase silica (30%).
At physiological ionic strength and pH (50 mM PIPES
(piperazine-N,N′-bis(2-ethanesulfonic acid), 100 mM KCl, KOH
* To whom correspondence should be addressed.
† Department of Pharmacology, University of California, San Diego.
§ Department of Chemistry and Biochemistry and Howard Hughes Medical
Institute, University of California, San Diego.
‡ Massachusetts Institute of Technology.
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(27) The name Zinpyr-1 was chosen to indicate the structural composition
of the ligand, containing four pyridyl groups, as well as its ability to “peer”
into the Zn2+ concentration of samples. Only Zn2+ and Cd2+ have been
observed to enhance Zinpyr-1 fluorescence, and other transition metal ions
can quench fluorescence, see Supporting Information.
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10.1021/ja000868p CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000