C O M M U N I C A T I O N S
Table 1. Chemical Properties of ZnAF-Rs with and without Zn2+ a
absorption maxima (nm)
emission maxima (nm)
fluorescence quantum yieldb
apparent K
d
dye
for Zn2+ (nM)
free base
Zn2+ complex
free base
Zn2+ complex
free base
Zn2+ complex
ZnAF-R1
ZnAF-R2
0.79
2.8
359
365
329
335
532
495
528
495
0.088
0.17
0.031
0.10
a All data were acquired in 100 mM HEPES buffer, pH 7.4. b Quantum yields were calculated using those of fluorescein (0.85) in 0.1 N NaOH as a
standard.21
due to differences in instrument efficiency and content of effective
dye. Therefore, this molecule should be useful for studies on the
biological functions of Zn2+
.
Acknowledgment. We thank Professor K. Yamaguchi and Dr.
S. Sakamoto in Chiba University for HRMS measurement. We also
thank Professor M. Iino in The University of Tokyo for donating
RAW 264.7. This work was supported in part by the Ministry of
Education, Science, Sports and Culture of Japan (Grants 11794026,
12470475, and 12557217 to T.N.; 13024217, 13558078, 1367232,
and 14045210 to K.K.), by the Mitsubishi Foundation, and by the
Research Foundation for Opt-Science and Technology.
Figure 1. Selectivity of ZnAF-R2. All data were obtained at pH 7.4 (100
mM HEPES buffer, I ) 0.1 (NaNO3)) and are expressed as fluorescence
ratio (335 nm/365 nm). Zn2+, Mn2+, Fe2+, Fe3+, Ni2+, Cd2+ (5 µM) were
added to 5 µM ZnAF-R2. Na+, Mg2+, K+, Ca2+ (5 mM) were added to 5
µM ZnAF-R2.
Supporting Information Available: Synthesis, experimental de-
tails, and characterization of ZnAF-Rs (PDF). This material is available
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Figure 2. Fluorescence ratiometric images (340 nm/380 nm) of zinc in
macrophages (RAW264.7) labeled with ZnAF-R2EE in PBS buffer, pH
7.4. (a) Bright-field transmission image. (b) Ratiometric image of (a). (c)
15 µM pyrithion (zinc ionophore) and 150 µM ZnSO4 were added to (b).
(d) 400 µM TPEN was added 15 min after the addition of pyrithione and
Zn2+
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Zn2+. Cu2+ and Co2+ formed complexes with ZnAF-R2 and strongly
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would have little influence in vivo, since they exist at very low
concentrations.22
To determine the cell permeability of ZnAF-R2, cultured
macrophages (RAW 264.7) were incubated with phosphate-buffered
saline (PBS) containing ZnAF-R2. The cells were not stained,
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membrane. Therefore, we prepared the ethyl ester derivative of
ZnAF-R2 (ZnAF-R2 EE).23 ZnAF-R2 EE is more lipophilic so that
it should permeate into the cell where it will be transformed into
ZnAF-R2 by esterase in the cytosol. Following incubation with PBS
and 10 µM ZnAF-R2 EE at 37 °C for 1.0 h, RAW 264.7 cells
were stained. The ratio of the fluorescences (340 nm/380 nm, using
the imaging system for fura-2) was increased immediately by the
addition of Zn2+ and 2-mercaptopyridine N-oxide (pyrithione),
which is a zinc-selective ionophore, to the medium, and the increase
was reversed by addition of TPEN. These data indicate that ZnAF-
R2 is a good candidate for a sensitive, practically useful Zn2+ probe
in biological applications (see Figure 2).
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(19) See Supporting Information.
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In conclusion, we have developed a new fluorescent probe for
Zn2+ that is membrane-permeable and has a high sensitivity.
Moreover, this molecule makes it possible to detect Zn2+ ratio-
metrically, thereby eliminating most or all of the possible variability
(22) Rae, T. D.; Schmidt, P. J.; Pufahl, R. A.; Culotta, V. C.; O’Halloran, T.
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(23) See Supporting Information.
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