A novel two-fluorophore approach to ratiometric sensing of Zn2+

Article abstract of DOI:10.1021/ja0364930

We present a small molecule ratiometric Zn²⁺-sensing system based on two fluorophores excited by visible light, a Zn²⁺-insensitive reporter fluorophore, coumarin 343, and a Zn²⁺-sensitive fluorescein-based compound, ZPA-1. The two fluorophores are linked by an ester to give Coumazin-1, a membrane-permeable, essentially nonfluorescent compound. Upon exposure to esterases, Coumazin-1 is hydrolyzed to its constituent fluorophores. Measurement of the ratio of coumarin emission at 488 nm (λ_exc = 445 nm) and comparison with ZPA-1 emission at 534 nm (λ_exc = 505 nm) affords information about the amount of sensor present as well as the amount of Zn²⁺ present. A generally applicable synthetic route to amide-functionalized ZP1 sensors is also described. The Zn²⁺-sensing properties of one member of this class are similar to those of the parent ZP1 sensor, with slightly tighter binding and lower background signal. Copyright

Full text of DOI:10.1021/ja0364930

Published on Web 08/27/2003
A Novel Two-Fluorophore Approach to Ratiometric Sensing of Zn2+
Carolyn C. Woodroofe and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received June 3, 2003; E-mail: lippard@lippard.mit.edu
Scheme 1. Synthesis of Coumazin-1a
The role of zinc in neurobiology has received significant
attention.1,2 Recent studies indicate that cellular Zn2+ levels are
tightly regulated.3 Understanding the biological functions of low
millimolar concentrations of loosely bound Zn2+ stored in vesicles
of hippocampal CA3 neurons4 is a subject of great interest. The
ability to track zinc release at the synapse in a time- and position-
sensitive manner requires a fast and quantitative detector such as
that provided by intensity-based fluorescent sensors.5,6 For zinc(II)
ions, it can be difficult to obtain a quantitative readout.7 To address
this problem, small-molecule ratiometric Zn2+ sensors have recently
been reported.8,9 These probes, however, are based on the Fura-2
and Indo-1 family of Ca2+ sensors and require UV excitation, which
can be damaging to cells and present problems with autofluores-
cence when applied in vitro. Available Zn2+ sensors with excitation
in the visible range are not ratiometric.10-13 A two-fluorophore
nanoparticle system has been described,14 but use of these particles
in cells requires microinjection. We report here an esterase-
mediated, small molecule, ratiometric, two-photon compatible
system comprising two fluorophores, both excited by visible light,
that should be of value in sensing Zn2+ in biological environments.
Coumazin-1, the synthesis of which is presented in Scheme 1,
is a prosensor containing a Zn2+-insensitive fluorescent coumarin
derivative covalently appended to a Zn2+-sensitive fluorescein
moiety. The latter is based on the recently reported Zn2+ sensor
Zinpyr-1 (hereafter, ZP1).11 The pyridinium salt of 2′,7′-dichloro-
fluorescein-3′,6′-diacetate-6-carboxylate was prepared in a manner
analogous to that of the 2′,7′-difluoro derivative.15 Activation with
oxalyl chloride followed by treatment with 4-aminobutanol afforded
the fluoresceinamido alcohol as depicted, which was coupled to
coumarin 343 under Mitsunobu conditions. Diacetate-protected
dichlorofluoresceins are viable substrates for Mannich reaction with
dipicolylamine and para-formaldehyde, obviating an intermediate
deprotection step. Coumazin-1 is membrane-permeable and es-
sentially nonfluorescent (Φ e 0.04). Treatment of the probe with
porcine liver esterase in vitro effects hydrolysis of the ester linkage,
yielding coumarin 343 and a PET-based Zn2+ sensor analogue of
ZP111 (Scheme 2).
a (a) Oxalyl chloride, DMF, -78 °C; (b) 4-aminobutanol; (c) PPh3,
DIAD, coumarin 343; (d) dipicolylamine, (CH2O)n, MeCN/H2O.
Scheme 2. Proposed Activation of Coumazin-1
Fluorescence spectra of esterase-treated micromolar Coumazin-1
solutions with coumarin excitation at 445 nm and ZP excitation at
505 nm before and after the addition of Zn2+ are shown in Figure
1. The ratio of ZP to coumarin emission intensity λ534/λ488 varies
from 0.5 in the absence of Zn2+ to 4.0 in the presence of a 2-fold
As indicated in Scheme 2, ester hydrolysis is presumed to
generate an amide-substituted ZP sensor. To assess its properties,
we prepared and characterized ZPA1 (Figure 2), which contains a
hydroxyethyl amide in the 6-position of the fluorescein. ZPA1 has
fluorescence and Zn2+-response properties similar to those of the
parent sensor ZP-111 (Table 1). After esterase processing, excitation
of the resulting coumarin product at 445 nm and measurement of
emission at 488 nm provides information about the concentration
of cleaved sensor. Excitation of the ZP fragment at 505 nm and
measurement of emission at 534 nm affords information about the
amount of Zn2+ present.
excess of Zn2+
.
The kinetics of ester hydrolysis were measured by monitoring
the increase in coumarin fluorescence over time. A fit to the
Michaelis-Menten model gave kcat ) 0.017(4) min-1 and kcat/Km
) 0.027(3) µmol-1 min-1 at 25 °C. Intracellular esterase hydrolysis
of esterified sensors is a common strategy used to trap the resulting
negatively charged carboxylate within the cell.16 Coumazin-1 is thus
transformed into a ratiometric sensing system by esterase processing
within the cell.
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J. AM. CHEM. SOC. 2003, 125, 11458-11459
10.1021/ja0364930 CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
Table 1. Comparison of Photophysical Properties of Zinpyr and
Coumazin Sensors
K (nM)c
pK a
2+
compound
Φfree
ΦZn
d
a
ZP1
ZPA1
Coumazin-1
0.38
0.87
0.7 ( 0.1
8.37
8.43
0.21
0.64
0.20 ( 0.02
0.01b,c
0.02b, 0.04c
0.25 ( 0.03d
a Protonation constant for benzylic amine responsible for quenching
fluorescence in the unbound state. b Excited in the coumarin absorption
region, ∼470 nm. c Excited in the fluorescein absorption region, ∼500 nm.
d Determined after treatment with and in the presence of the esterase.
excitation, based upon the esterase-mediated cleavage of a member
of the Zinpyr family of Zn2+ sensors functionalized with a Zn2+
-
insensitive reporter fluorophore. This strategy is generally applicable
as a modification to existing sensors for all biological ana-
lytes.
Figure 1. Fluorescence spectra of an esterase-treated solution of Cou-
mazin-1 (2 µM) excited at 445 nm (circles) and 505 nm (squares) before
(solid) and after (open) addition of excess ZnCl2 (20 µM).
Acknowledgment. This work was supported by the McKnight
Foundation for the Neurosciences and NIH grant GM65519. We
thank Katie R. Barnes and Olga Burenkova for assistance with cell
studies. C.C.W. acknowledges a predoctoral fellowship from Merck/
MIT. Spectroscopic equipment was maintained with support from
NIH grant 1S10RR13886-01 and NSF grants CHE-9808063,
DBI9729592, and CHE9808061.
Supporting Information Available: Syntheses and experimental
procedures (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 2. Structure of ZPA1.
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Figure 3. Phase contrast (left) and fluorescence (middle, right) microscopy
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To test these expectations and to determine the extent of
colocalizaton of the two fluorophores, we incubated HeLa cells
with 5 µM Coumazin-1. Subsequent imaging with a dual-filter
fluorescence microscope afforded the images shown in Figure 3.
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In conclusion, we describe a versatile synthetic route to amide-
containing ZP1 sensors. We applied this methodology to produce
a small-molecule ratiometric sensing system for Zn2+ with visible
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