C O M M U N I C A T I O N S
Figure 3. Bright-field transmission (a) and ratiometric images (b) of CMFâ-
gal-injected LacZ-positive HEK293 cells at 300, 800, and 1300 s after
microinjection. See Supporting Information for details.
Figure 1. The emission spectra of a 1.0 µM solution of CMFâ-gal after
the addition of â-galactosidase (11.6 µg‚mL-1) in phosphate buffer (10 mM,
pH 7.4) at 37 °C. The spectra were measured at every 4 min after the
addition of â-galactosidase, λex ) 400 nm.
synthesized a â-galactosidase labeling probe, CMFâ-gal, the
labeling reaction of which can be monitored in terms of the change
of fluorescence wavelength. This is the first report of a protein
labeling probe which features a change of fluorescence wavelength
upon reaction, allowing the labeled protein to be detected even in
the presence of unreacted probe. The general probe design allows
a wide tolerance in the selection of fluorophore pairs as well as
protective groups for various enzymes. In applying this system to
the other target enzymes, one possible limitation is that the
feasibility of the labeling may depend on the structure of the protein,
especially on the location of nucleophilic amino acids to be
labeled.9,10 Though, in future work, we think it would be possible
to develop a range of probe and target enzyme pairs that serve as
powerful tools in the study of protein dynamics in living cells.
Acknowledgment. We thank Haruhiko Bito and Hajime Fujii
for technical support with SDS-PAGE. This work was financially
supported by the Ministry of Education, Culture, Sports, Science
and Technology of Japan (Grants for The Advanced and In-
novational Research Program in Life Sciences, 16370071 and
16659003 to T.N., 15681012, 17651119, 17048006, 17035019, and
17036012 to K.K.). T.N. was also supported by the Hoh-ansha
Foundation. K.K. was also supported by the Sankyo Foundation,
by the Kanagawa Academy of Science, and by the Suzuken
Memorial Foundation.
Figure 2. The result of SDS-PAGE (see Supporting Information for details
of sample preparation). Left: image of Coomassie brilliant blue-stained
gel. Right: fluorescence image of the gel under illumination at 365 nm
from a UV lamp.
to be >93%. When â-galactosidase was added to the solution, a
rapid increase in the emission of 7-hydroxycoumarin and a
corresponding rapid decrease in the emission of fluorescein were
observed. Monitoring of the increase of the ratio of fluorescence
intensities at 460 and 515 nm allowed accurate estimation of the
â-galactosidase concentration (see Supporting Information). After
fluorescence change of reaction mixture had ceased, â-galactosidase
was purified and its spectrum was measured to confirm that CMFâ-
gal had indeed acted as a labeling probe of â-galactosidase. As
shown in the Supporting Information, purified â-galactosidase
exhibited the characteristic absorbance and fluorescence spectra of
7-hydroxycoumarin. These results were the same as that obtained
with a control compound, CMfluorideâ-gal. In contrast, no labeling
occurred when the probe was mixed with a control protein,
â-glucosidase, instead of â-galactosidase. The labeled protein could
also be detected with SDS-PAGE. As shown in Figure 2,
fluorescence of 7-hydroxycoumarin was observed at the spot of
â-galactosidase labeled with the probe, while it was not observed
at the spot of â-glucosidase.
Finally, CMFâ-gal was used for ratiometric imaging in LacZ-
positive or LacZ-negative cells to confirm that it can detect
â-galactosidase expression in living cells (Figure 3). After micro-
injection of the probe, slight leakage occurred, but the increase in
the emission of 7-hydroxycoumarin was clearly observed in LacZ-
positive cells, while it was not apparent in LacZ-negative cells (see
Supporting Information).
In conclusion, we have established a novel design strategy for
quinone methide chemistry-based protein labeling probes, with
carbamate as a leaving group. On the basis of this design, we
Supporting Information Available: Synthesis, experimental de-
tails, and characterization of CMFâ-gal and CMfluorideâ-gal, and
additional experiments. This material is available free of charge via
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