Angewandte
Chemie
cence intensity of FCANB was weak (Ff = 0.04) and was
augmented by the binding of FCANB to the PYP tag (Ff =
0.47); its fluorescence change was 15-fold and thus larger than
that of FCATP. Neither of the probes displayed a time-
dependent alteration of the fluorescence intensity in the
absence of PYP tag (Figure S2 in the Supporting Informa-
tion). These results demonstrate that both FCATP and
FCANB are fluorogenic probes for labeling PYP tag. More-
over, they show that the nitrobenzene moiety in FCANB
contributes to the quenching efficiency by lowering the
fluorescence quantum yield and extinction coefficient of the
fluorophore under these experimental conditions (Table 1).
The absorption spectra of free FCATP and FCANB show that
the maximum wavelength was shifted by 7 nm in comparison
to that of the fluorescein derivative without a ligand or
nitrobenzene moiety (Figure S3 in the Supporting Informa-
tion). This spectral change strongly suggests that intramolec-
ular association occurs between the fluorophore and the
ligand or nitrobenzene, and this association could cause
fluorescence quenching for both probes. This type of quench-
ing mechanism has also been reported, and it has been shown
that fluorescence quenching is triggered by the close contact
of intramolecular fluorophores.[8,15]
The kinetic analyses of protein labeling were carried out
by monitoring the increase in the fluorescence intensity of the
probes. The time required for 50% labeling, t1/2, was
estimated using the labeling reactions, in which the concen-
trations of the probes and proteins were 8 mm (Figure 2b,
Figure S2 in the Supporting Information). FCANB has the
shortest t1/2 value (ca. 15 min), followed by FCATP (t1/2 ca.
78 min) and FCTP (t1/2 > 470 min), thus demonstrating that
FCANB binds to the PYP tag most rapidly. In further kinetic
analyses, second-order rate constants were also calculated
(Table 1, Figure S4 in the Supporting Information). Consis-
tent with the t1/2 values, the k2 values of FCATP and FCANB
are 10-fold and 110-fold higher than that of FCTP, respec-
tively. These kinetic data suggest that the introduction of the
4-hydroxycinnamic acid ligand in probes FCATP and
FCANB leads to the fast protein labeling. There is a possibility
that the PYP tag could intrinsically bind to the 4-hydroxycin-
namic acid ligand more rapidly than to the coumarin ligand.
However, this possibility was excluded, because it was found
that the PYP tag binds to both ligands, neither of which
contains the fluorophore, with almost the same kinetics
(Figure S5 in the Supporting Information). Therefore, it is
more likely that the strength of intramolecular interaction or
the structure of the intramolecular complex between the
fluorophore and ligand moieties affects the protein-labeling
kinetics of both probes FCANB and FCATP. As well as the 4-
hydroxycinnamic acid ligand, nitrobenzene also gave a pro-
motional effect on the protein labeling kinetics of FCANB, as
expected. This result suggests that the fluorophore interacts
more favorably with the nitrobenzene rather than with the
ligand moiety, and thereby the steric hindrance around the
ligand is diminished.
kidney (HEK293T) cells (Figure S6 in the Supporting Infor-
mation), and each of the probes was incubated with the cells
for 30 min. After washing the cells to remove free probes,
fluorescence images were taken using confocal laser-scanning
microscopy (Figure 3a, Figure S7a in the Supporting Infor-
Figure 3. Live-cell imaging of PYP-tagged EGFR on cell surfaces with
the probe FCANB (5 mm) a) with or b) without washing procedures.
Fluorescence images and their overlays with phase contrast images
are shown in the left and right of each panel, respectively. Scale bars:
10 mm.
mation). Clear fluorescence was observed along the plasma
membrane in the cells treated with either FCATP or FCANB.
No fluorescence was detected in cells expressing EGFR
without PYP tag. These results indicate that both probes
specifically label the PYP–EGFR fusion protein on the cell
surface. We also tried to label PYP-tagged proteins inside
cells with the probes. This attempt, however, failed, because
the probes were not cell-permeable. Taking advantage of the
fluorogenic properties of the probes, direct imaging of cell-
surface proteins without washing was performed immediately
after the labeling reaction (Figure 3b, Figure S7b). As in
images obtained when the washing procedure was used,
distinct fluorescence was detected only on the surface of the
cells expressing the PYP–EGFR fusion protein, while the
fluorescence of the free probe was not seen in the media or in
other parts of cells. Nonspecific labeling was also confirmed to
be absent in cells that do not express the PYP–EGFR fusion
protein. Importantly, specific imaging of proteins was accom-
plished by utilizing these fluorogenic probes without washing.
In summary, we have developed fluorogenic probes,
FCATP and FCANB, for labeling PYP tag. Kinetic properties
of the probes were significantly improved compared to the
previous probe, FCTP. In particular, FCANB binds to the
PYP tag 110 times more rapidly than FCTP. This acceleration
effect is induced by the introduction of both the cinnamic acid
ligand and the nitrobenzene quencher into the probe
structure. A possible reason for this effect is that the
fluorophore preferably interacts with the nitrobenzene
instead of the ligand and that thereby the steric hindrance
around the ligand is reduced. The kinetic enhancement and
fluorogenicity of the probes enabled the specific labeling of
PYP-tagged proteins on the cell surface with a procedure that
does not require washing steps. The most notable point is that
the PYP tag is the smallest protein among existing protein
tags that can be covalently labeled by small fluorogenic
compounds without the requirement of washing cells. This no-
wash labeling system combined with the small PYP tag offers
an attractive tool for the imaging of rapid movement and
Finally, live-cell imaging was conducted using the probes.
Epidermal growth factor receptor (EGFR) fused with the
PYP tag at the N-terminal extracellular domain (PYP–
EGFR) was expressed on the surface of human embryonic
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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