Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes

Article abstract of DOI:10.1021/ja304239g

Visualization of tumor-specific protein biomarkers on cell membranes has the potential to contribute greatly to basic biological research and therapeutic applications. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore in test tube settings. This method is based on recognition-driven disassembly of the nanoprobes, which induces a clear turn-on fluorescent signal. In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-assembling turn-on probes.

Full text of DOI:10.1021/ja304239g

Article
pubs.acs.org/JACS
Specific Cell Surface Protein Imaging by Extended Self-Assembling
Fluorescent Turn-on Nanoprobes
Keigo Mizusawa,† Yousuke Takaoka,† and Itaru Hamachi*,†,‡
†Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan
‡Japan Science and Technology Agency (JST), CREST, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
S
* Supporting Information
ABSTRACT: Visualization of tumor-specific protein bio-
markers on cell membranes has the potential to contribute
greatly to basic biological research and therapeutic applica-
tions. We recently reported a unique supramolecular strategy
for specific protein detection using self-assembling fluorescent
nanoprobes consisting of a hydrophilic protein ligand and a
hydrophobic BODIPY fluorophore in test tube settings. This
method is based on recognition-driven disassembly of the
nanoprobes, which induces a clear turn-on fluorescent signal.
In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as
fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available
fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate
receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific
biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-
based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-
assembling turn-on probes.
of a more general switching strategy for protein-specific
fluorescent probes is highly desired.
INTRODUCTION
■
Tumor-specific or tissue-selective protein biomarkers overex-
pressed on cell surfaces are important targets for basic
biological research studies and therapeutic applications.1 In
most cases, antibodies appended with fluorescent molecules are
used to visualize these endogenous biomarkers on live cells.
However, they are always fluorescent, which inevitably requires
a lot of washing operations for accurate molecular imaging on
cells. Introduction of a switching mechanism (of fluorescent
intensity and/or wavelength change only responsive to the
target protein) should greately help to solve this problem.2
Recently, Urano and co-workers achieved highly specific in vivo
cancer visualization using an antibody conjugated with novel
acidic pH-activatable fluorescent probes.3 Although undoubt-
edly valuable, such antibody-based probes possess several
inherent problems, that is, besides their high cost, only a few
antibodies can be prepared in large amounts, and site-specific,
quantitative conjugation without loss of the antibody specificity
remains difficult. On the other hand, small-molecule-based
switchable fluorescent probes for the specific target proteins are
particularly promising for imaging biomarkers, because they
allow rapid, sensitive, and selective detection. The most
prevalent probes are fluorogenic substrates that monitor the
corresponding enzyme activity. However, this strategy can only
be applied to particular enzymes (e.g., proteases or
glycosidases),4,5 not nonenzymatic proteins including the
myriad of receptors in living systems. Clearly, establishment
Recently, several supramolecular approaches using amphi-
philic polymers or dendrons for protein detection have been
reported.6,7 Although these nanomaterials are useful for protein
sensing and profiling in a test tube context, they cannot be used
for protein sensing under the crude cellular conditions because
of their low selectivity or sensitivity to date. We also recently
developed a disassembly-driven turn-on nanoprobe for specific
protein detection.8,9 The ligand-tethered fluorophores exhibited
unique self-assembling features and showed the clear turn-on
fluorescence change with high protein sensitivity.9 However,
only the hydrophobic fluorophore BODIPY was valid in our
fluorescent probes, which greatly hampered their live cell
application. Herein we extend the range of usable fluorophores
to the more hydrophilic ones such as fluorescein or rhodamine
by introducing a hydrophobic module near the fluorophore in
the turn-on nanoprobe. Using the newly synthesized self-
assembling fluorescent nanoprobes, we succeeded in imaging
cancer-specific biomarkers such as the folate receptor (FR) and
transmembrane-type carbonic anhydrases (CA) under a live cell
setting (Scheme 1). Furthermore, a cell-based inhibitor
screening system for CAs under hypoxic live cell conditions
was successfully constructed.
Received: May 2, 2012
Published: July 18, 2012
© 2012 American Chemical Society
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Scheme 1. Schematic Illustration of a Self-Assembling Turn-
on Fluorescent Probe for Cell Surface Protein Imaging
RESULTS AND DISCUSSION
■
Self-Assembling BODIPY-Based Fluorescent Probes
toward DHFR Detection and FR Imaging. FR, a
membrane-bound protein that is a potential biomarker because
of its overexpression among malignant tumor cells, was the first
target in a proof-of-principle experiment.10 Using our previous
design strategy, we newly prepared a turn-on fluorescent probe
1 to image FR. This probe consists of three modules: (i) a
hydrophilic ligand specific to a protein of interest (methotrex-
ate (MTX) as the ligand with an affinity for FR11), (ii) a
fluorescent BODIPY as both a detection modality and a
hydrophobic core, and (iii) a relatively hydrophobic linker
group to connect these two modules and control the
hydrophilic/hydrophobic balance (Figure 1a).
To test the turn-on property of probe 1 in vitro, we initially
employed purified dihydrofolate reductase (DHFR) as a water-
soluble model protein which can bind the MTX ligand in the
same way as FR.12 As shown in Figure 1b, the emission was
extremely weak when 1 was dissolved in aqueous solution, but
dramatically increased (by 23 times) upon the addition of
DHFR. That is, probe 1 was an ideal off/on type of fluorescent
Figure 1. MTX-type BODIPY probe 1 for DHFR detection in the test tube and for FR imaging on live cell surfaces. (a) Chemical structure of probe
1. (b) Fluorescence spectral changes of probe 1 (15 μM) upon addition of DHFR (0−45 μM) in test tube (λex = 480 nm). (c, d) Fluorescence (left,
FL) and differential interference images (right, DIC) of (c) KB cells or (d) HEK293T cells treated with probe 1 (15 μM). Scale bars, 40 μm.
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Figure 2. Chemical structures of self-assembling probes based on hydrophilic fluorophores for FR and CA imaging: 2−5 for FR and 6−8 for hCAs.
probe for DHFR at least in test tube settings. We next applied 1
to live cell imaging of KB cells overexpressing FR.13
Unfortunately, when a solution of 1 was added to the cultured
KB cells, strong emission was observed inside the cells as well
as on the cell surfaces in confocal laser scanning microscopy
(CLSM) images (Figure 1c). The strong emission was also
observed by using 1 even in the presence of an excess amount
of folate, which is a competitive ligand for FR (Figure S1,
Supporting Information [SI]). In addition, similar bright
fluorescence was detected when 1 was mixed with HEK293T
cells that did not express FR (Figure 1d).14 It is clear that probe
1 could not selectively image FR, mainly due to its nonspecific
binding to cell membranes and its cell permeability. These
results indicate that both the turn-on fluorescent property and
efficient suppression of nonspecific binding to cells should be
considered for application of the self-assembled nanoprobes in
live cell imaging.
Design of Fluorescein-Based Self-Assembling Turn-on
Probes by Introduction of a Hydrophobic Module. To
suppress nonspecific binding to live cell membranes, the
fluorophore module was changed from the rather hydrophobic
BODIPY to an anionic fluorescein (Fl). Similar to 1, Fl-type
probe 2 was composed of a simple sulfonamide linker for
connecting MTX ligand and Fl (Figure 2). However, 2 alone
showed a relatively large fluorescence (its fluorescence
quantum yield (Φ) was 0.11), and thus the fluorescence
enhancement induced by addition of DHFR was only 3.5-fold
(Φ = 0.36) (Figure S2c, [SI]). That is, the inherent
fluorescence of 2 was not efficiently quenched, probably
because of the strong hydrophilicity of the Fl group, and thus
this probe was identified as an always-on type of probe. To
achieve more effective quenching, hydrophobic dipeptide (di-
(4-trifluoromethyl-phenylalanine), [F(CF3)]2) was introduced
as an additional hydrophobic module adjacent to the
fluorophore in the Fl-type probe (probe 3). As expected, the
emission of 3 dissolved in aqueous solution was sufficiently
weak (Φ = 0.03), and dramatically increased (by 10 times)
upon addition of DHFR (Φ = 0.25, Figure 3b and c). Atomic
force microscopy (AFM) images revealed the formation of
spherical or oval aggregates of 3 with diameters ranging from
10 to 50 nm (Figure 3d). In dynamic light scattering (DLS)
measurements, a buffer solution containing only 3 showed
aggregates with a mean diameter of 30 nm (Figure 3e), whereas
negligible scattering intensity was observed after addition of
DHFR to this solution. These results indicate that probe 3
works as an ideal off/on type of probe driven by the
recognition-triggered disassembly in the test tube. On the
other hand, probe 4 containing a more hydrophobic
tetrapeptide (tetra-phenylglycine, (Phg)4, Figure 2) as the
hydrophobic module exhibited almost no fluorescence both in
the absence (Φ = 0.02) and presence of DHFR (Φ = 0.04,
Figure 3c and Figure S2d [SI]), which classified 4 into an
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Figure 3. (a) UV−vis absorption spectral changes of probe 3 (10 μM) upon addition of DHFR (0−20 μM). (b) Fluorescence spectral changes of
■
◆
,
●
probe 3 (10 μM) upon addition of DHFR (0−20 μM) (λex = 450 nm). (c) The fluorescence titration curves of 2 ( , red), 3 ( , black), or 4 (
blue) following addition of DHFR in test tube experiments. (d) AFM image of probe 3 only (10 μM, scale bar 200 nm). (e) DLS analysis of probe 3
only (10 μM). (f) Photographs of probe 3 (10 μM) in the absence (left) and presence (middle) of DHFR (10 μM), and after addition of MTX (100
μM) to the solution of 3 and DHFR (right). These images were obtained under UV excitation (λex = 365 nm). All experiments were performed in 50
mM HEPES buffer (pH 7.2, 150 mM NaCl).
always-off type of probe (Table 1 and Figure S3 [SI]). Given all
results in the Fl-type probes, it is clear that control of the
hydrophobicity in the tail group containing hydrophilic
fluorescein is critical for designing an ideal off/on type of
fluorescent probe. In our previous study on 19F NMR/MRI
probes, we reported that the stability of aggregates was
controlled by the hydrophobicity/hydrophilicity balance,
which was crucial for an off/on 19F NMR response.8b The
present findings imply that a similar strategy is valid for
fluorescence signal switching in the self-assembling probes. In
the case of tetramethyl rhodamine (TMR) as another
hydrophilic fluorophore, we indeed successfully developed the
off/on type of probe 5 by introduction of phenylalanine as a
hydrophobic module (Figures 2 and 4). Probe 5 (10 μM)
exhibited a 37-fold enhancement of fluorescence from TMR at
584 nm upon addition of one equivalent of DHFR. This
demonstrated that incorporation of a hydrophobic unit adjacent
to the hydrophilic fluorophore is a general strategy to produce
turn-on self-assembling fluorescent nanoprobes.
Table 1. Hydrophobic Modules and Response Patterns of
a
Fluorescein-Type Probes
hydrophobic
Φ without
Φ with
response
patterns
b
b
probe target
module
protein
protein
MTX−Fluorescein-Type of Turn-on Probe for Fluo-
rescent FR Imaging on Live Cell Surfaces. With the Fl-type
of turn-on probe 3 in hand, we again set out to image FR on
live cells. When probe 3 was added to KB cells, strong
fluorescence in the CLSM image was observed only from the
cell surface without requiring any washing operations (Figure
5a). The intensity of fluorescence on the cell surface decreased
dramatically upon addition of folate without any change in the
intensity of fluorescence in the extracellular region (Figure 5b).
These data clearly indicate that turn-on fluorescence switching
took place on live KB cell surface, and that this switching is
reversible and controlled by the recognition of FR by the ligand
moiety of 3. In addition, probe 3 is not cell permeable, allowing
FR to be visualized specifically on the cell surface. On the other
hand, when DHFR was added to the solution containing KB
2
3
4
6
FR/
DHFR
−
0.111
0.032
0.021
0.042
0.356
0.251
0.040
0.342
always-on
FR/
DHFR
[F(CF3)]2
(Phg)4
off/on
FR/
DHFR
always-off
off/on
hCAs
Ahep-
[F(CF3)]2
7
8
hCAs
hCAs
−
0.457
0.057
0.423
0.078
always-on
[F(CF3)]3
always-off
a
Quantum yields were calculated by using fluorescein as a reference
b
standard in test tube settings. FR: folate receptor; DHFR:
dihydrofolate reductase; hCAs: human carbonic anhydrases; F(CF3):
4-trifluoromethyl-phenylalanine; Phg: phenylglycine; Ahep: 2-amino-
heptanoic acid.
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Figure 4. (a) UV−vis absorption spectra changes of TMR-type probe 5 (10 μM) upon addition of DHFR (0−20 μM). (b) Fluorescence spectra
changes of probe 5 (10 μM) upon addition of DHFR (0−20 μM) (λex = 480 nm). (c) Photographs of probe 5 (10 μM) in the absence (left) and
presence (middle) of DHFR (10 μM), and after addition of MTX (100 μM) to the solution of 5 and DHFR (right). These images were obtained
under UV excitation (λex = 365 nm). All experiments were performed in 50 mM HEPES buffer (pH 7.2, 150 mM NaCl).
cells stained with 3, the strong emission from the extracellular
region was observed (Figure 5c). This data explicitly reveal that
an excess amount of residual probe 3 forms self-assembled
aggregates in solution, which retained the fluorescence signal-
off state. Besides, no substantial fluorescence was observed
from HEK293T cells with 3 (Figure 5d). All of the results
demonstrate that the probe 3 is capable of specific fluorescent
imaging of endogenous FR in a turn-on manner, unlike the
original BODIPY-type probe 1. Importantly, probe 3 efficiently
suppressed the background signal from the extracellular region
even under cellular conditions. By contrast, the higher
background signals were observed from the extracellular region
using the always-on-type probe 2, whereas no detectable signals
were observed with the use of the always-off type probe 4
(Figure S4, [SI]). These data supported that an ideal off/on
type of probe could be synthesized by control of the self-
assembling properties which would enable us to perform rapid
live cell imaging of FR without requiring any extra operations.
Development of Sulfonamide−Fluorescein-Type
Probe for Fluorescent Transmembrane-Type CA Imag-
ing under Hypoxic Live Cell Conditions. Benefiting from
the modular design, a self-assembling nanoprobe was
constructed for fluorescent live cell imaging of another
membrane protein. It has been reported that transmembrane-
type human carbonic anhydrases (hCA IX and XII) are
substantially overexpressed in response to the hypoxia inducible
factor (HIF-1) in tumors.15 Recent studies have also shown
that CA IX is involved in tumorigenesis through pH regulation,
and manipulation of cell proliferation and cell adhesion, making
it a valuable target for cancer diagnostics and treatment.16,17 On
the basis of the same strategy as for the FR probes, off/on type
probe 6 containing a benzenesulfonamide (SA) ligand for
selective binding to the CA family was developed. In this case,
hydrophobic tripeptide (2-aminoheptanoic acid and di-(4-
trifluoromethyl-phenylalanine), Ahep-[F(CF3)]2, Figure 2), as
the hydrophobic module was found to produce an ideal off/on
type of probe (9-fold increase in fluorescent intensity, shown in
a and b of Figure 6 and Figure S5 [SI]) in test tube experiments
by evaluating the fluorescence response to hCA II, a soluble
model of hCA IX. As in the case of the FR probes, always-on
and always-off probes were also found among these CA probe
candidates (Figure 2, Table 1, and Figure S6 [SI]) by varying
the hydrophobic tail group.
Subsequently, imaging of the CA family on live cell surfaces
was conducted using probe 6. The A549 cell line was chosen,
owing to its relatively high endogenous expression of hCA IX
or XII under hypoxic conditions.15a Three parallel sets of A549
cells were respectively cultured for 24 h under hypoxic (<0.1%
O2), hypoxic-mimetic (in the presence of deferoxamine
mesylate (DFO)), or normoxic conditions (20% O2). When
6 was added to A549 cells cultured under the hypoxic
conditions, strong emission was observed from the cell surface
without any washing operations (Figure 6c).18 The intensity of
fluorescence decreased dramatically upon addition of ethox-
azolamide (EZA), a strong inhibitor for CAs (Figure 6d).
Similar clear fluorescent images were obtained from the cells
incubated in the presence of DFO (Figure 6e), whereas
significantly weaker emission was observed from A549 cell
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Figure 5. (a, b) Fluorescence images of KB cells treated with probe 3 (20 μM) in the (a) absence or (b) presence of folate (20 μM). (c)
Fluorescence image of KB cells treated with probe 3 (20 μM) with purified DHFR (5 μM). (d) Fluorescence image of HEK293T cells treated with
probe 3 (20 μM). Scale bars, 40 μm. Fluorescence imaging experiments were performed 1 min after the addition of the probe solution to the cells.
surfaces under normoxia (Figure 6f). In addition, negligible
fluorescence was observed from HEK293T cells not expressing
hCA IX (Figure S7, SI). Given all the data, we concluded that
the probe 6 is capable of sensing not only the expression of the
transmembrane-type hCA family but also the hypoxic environ-
mental change of cancer cells based on the level of
overexpression of the biomarkers.
Finally, imaging-based drug screening for hypoxia-inducible
CAs was performed on live A549 cells using fluorescein-type
probe 6. After staining with 6 (10 μM), the A549 cells were
mixed with several CA inhibitors, and changes in fluorescence
images were monitored by CLSM without any washing
operations. When the strong inhibitor EZA (100 nM) was
added to the culture medium, a significant decrease in
fluorescent intensity at the cell surface was induced with
typical saturation behavior (Figure 7a). In the case of moderate
inhibitors AAZ and SA-Py, the change in the CLSM image was
saturated at 5 and 100 μM, respectively (b and c of Figure 7
and Figure S8, SI). In contrast, a very small change was
observed upon addition of a higher concentration of 4-
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Figure 6. (a, b) Turn-on detection of hCAII with probe 6 in test tube settings. (a) Fluorescent spectral changes of probe 6 (10 μM) upon addition
of hCAII (0−20 μM). (b) Photographs of probe 6 (10 μM) in the absence (left) and presence (middle) of hCAII (10 μM), and after addition of
EZA (100 μM) to the solution of 6 and hCAII (right). (c, d) Transmembrane-type hCA imaging of A549 cells using probe 6 (10 μM) (c) without or
(d) with EZA (100 μM) cultured under hypoxic conditions. (e) Transmembrane-type hCA imaging of DFO-treated A549 cells using probe 6 (10
μM). (f) Transmembrane-type hCA imaging of A549 cells using probe 6 (10 μM) cultured under normoxic conditions. Scale bars, 40 μm.
Fluorescence imaging experiments were performed 1 min after the addition of the probe solution to the cells.
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Figure 7. Imaging-based CA inhibitor cell-based assay with probe 6. (a−d) Fluorescence images of hypoxia-cultured A549 cells treated with probe 6
(10 μM) in the presence of CA inhibitors [(a) EZA (100 nM), (b) AAZ (100 nM), (c) AAZ (5 μM), and (d) ABS (200 μM)). Scale bars, 40 μm.
(e) Chemical structures of inhibitors for transmembrane-type CA used in this study. EZA is 6-ethoxy-2-benzothiazolesulfonamide, AAZ is
acetazolamide, SA-Py is trimethylpyridinium derivative of benzenesulfonamide, and ABS is 4-aminobenzenesulfonic acid. (f) Fluorescence titration
●
○
profile of the relative change in fluorescence intensity (I/I0 − 1) from the cell surface region vs inhibitor concentration for EZA ( , red), AAZ ( ,
□
blue), SA-Py (×, green), and noninhibitor ABS ( , black). Fluorescence intensities were evaluated at five regions in each dish, and the experiments
were performed in triplicate to obtain mean and standard deviation values (shown as error bars).
aminobenzenesulfonic acid (ABS), which is not a ligand for the
CA family (Figure 7d). From the titration curves of these live-
cell-based inhibitor assays, IC50 values of each inhibitor were
determined to be 2.3 × 10−8 M (EZA), 2.4 × 10−7 M (AAZ),
and 2.2 × 10−5 M (SA-Py) (Figure 7e and f). On the other
hand, the literature values (Ki) for hCA IX were reported to be
CONCLUSION
■
In summary, we have developed self-assembling turn-on
fluorescent nanoprobes that can be used for fluorescent
imaging of protein biomarkers under live cell contexts, as
well as in test tube settings. The design of these nanoprobes
was based on recognition-driven disassembly of a ligand-
tethered fluorophore, by newly introducing a hydrophobic
module to finely tune the aggregation properties of the probe.
This greatly enhanced the flexibility of the probe design. The
probes allowed specific visualization of overexpressed FR and
hypoxia-inducible CA on cancer cells, both of which are tumor-
specific biomarkers, without needing any washing operations.
Moreover, an extended nanoprobe was successfully used in a
cell-based inhibitor assay for CA on the surface of live cells.
This highlights the supramolecular approach for bioimaging
under live cell conditions. Nowadays, many small-molecule
drug candidates toward various membrane-bound proteins have
been reported. By using such drugs, our approach using the
3.4 × 10−8 M (EZA), 2.5 × 10−8 M (AAZ), and 1.4 × 10−8
M
(SA-Py),17c the values of which were obtained by the purified
system using the catalytic domain of hCA IX. While the values
for EZA were almost same in the order, those for AAZ and SA-
Py were apparently different. This discrepancy could be caused
by the differences in these two assay systems. For example, the
inhibitive capacities of AAZ and SA-Py may be decreased by the
cell penetration or nonspecific binding to the cell membrane. It
is conceivable that the cell-based inhibitor assays by the self-
assembling probes allow us to evaluate the actual potencies in
live cell settings.
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units/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2 and
95% air. The cells were plated at a density of 2.0 × 105 cells per a 35-
mm glass-bottomed dish and cultured for 24 h at 37 °C in air with 5%
CO2. After the medium exchange, the cells were cultured for 24 h at 37
°C under hypoxic conditions (<0.1% O2) generated with an
AnaeroPack (Mitsubishi Gas Chemical Company. Inc.) and a
rectangular jar. For control experiments, parallel normoxic dishes
were incubated in DMEM with or without DFO (200 μM) for 24 h at
37 °C in air with 5% CO2. The cells were rinsed with 1 × PBS buffer,
and then treated with the probe 6 (10 μM, <1% DMSO (v/v)] or the
probe 6 (10 μM) and EZA (100 μM) (<1% DMSO (v/v)] in 1 × PBS
buffer (1 mL). Fluorescence imaging was carried out 1 min after
staining without performing any washing operations.
Inhibitor Assay for Membrane-Bound hCA Family on A549
Cells. The concentration of probe 6 was fixed at 10 μM in each
experiment. After adding probe 6 to the A549 cells cultured under
hypoxic conditions as described above, a stock solution of each
inhibitor was added to the cells stained with the probe and
fluorescence imaging was carried out without performing any washing
operations. To determine the fluorescence intensity on the cell
membrane, CLSM images were analyzed with ImageJ 1.44 on a
Macintosh PC.
turn-on nanoprobes should be applied to the live-cell imaging
of various membrane-bound proteins. We believe that the
present progress in our turn-on nanoprobes should facilitate the
development of various target-specific signal-switchable probes
for use in cells or in vivo.
EXPERIMENTAL SECTION
■
Synthesis. Synthetic procedures used to prepare compounds 1−6
and 8, and characterizations of these compounds are described in the
Supporting Information. Compound 7 was prepared as previously
reported.17a
General Materials and Methods. Purified human carbonic
anhydrase II (CAII) was purchased from Sigma, and Escherichia coli
DHFR was prepared and purified as previously reported.19 BODIPY
558/568 SE, 5-carboxyfluorescein succinimidyl ester, and 5-carbox-
ytetramethylrhodamine succinimidyl ester were purchased from
Invitrogen/Molecular Probes. Other chemical reagents and solvents
were purchased from commercial chemical suppliers (Sigma, Aldrich,
TCI, Wako, or Watanabe Chemical Industries) and used without
further purification. 1H NMR spectra were recorded on a Varian
Mercury-400 spectrometer (400 MHz). High-resolution electrospray
ionization quadrupole Fourier transform mass spectrometry (HR-ESI-
MS) spectra were performed on a Bruker apex-ultra (7 T) mass
spectrometer. Cell imaging was performed with a confocal laser
scanning microscope (CLSM, Olympus, FV1000, IX81) equipped with
a 60×, NA = 1.35 oil objective lens. Fluorescence images were
acquired using the 488 nm line of an argon laser to excite fluorescein
(emission, 500−600 nm) and the 543 nm line of a HeNe Green laser
to excite BODIPY 558/568 (emission, 550−650 nm). Measurements
were carried out 1 min after staining without performing any washing
operations.
UV−Visible Absorption and Fluorescence Spectroscopic
Analyses. All probes were dissolved in dimethyl sulfoxide (DMSO)
to generate stock solutions. The concentrations of probe 1 and 5 were
determined by their absorbance (at 559 nm for 1 and 543 nm for 5) in
methanol using a molar extinction coefficient of 97,000 M−1 cm−1 for
1 and 92,000 M−1 cm−1 for 5.20 The concentration of fluorescein-type
probes (2−4 and 6−8) were determined by their absorbance at 494
nm in 0.1 N NaOH aq using a molar extinction coefficient of 75,000
M−1 cm−1.20 hCAII was dissolved in 0.1 M Tris·HCl, and 0.1 M
Na2SO4 buffer (pH 7.5), and DHFR was dissolved in 1 × PBS buffer
(pH 7.2). The concentrations of these proteins were determined by
their absorbance at 280 nm using a molar extinction coefficient of
54,000 M−1 cm−1 for hCAII,21 and 31,100 M−1 cm−1 for DHFR,22
allowing stock solutions of known concentrations to be prepared. All
experiments were performed at 25 °C. UV−visible absorption spectra
were recorded on a Shimadzu UV−visible 2550 spectrometer.
Fluorescence spectra were measured using a Perkin-Elmer LS55
fluorescent spectrometer. Absorption and fluorescence measurements
were performed 1 h after adding the protein to each probe solution
(10 μM). The fluorescence quantum yields of probes 2−4 and 6−8
were calculated by using fluorescein (Φ = 0.85 in 0.1 N NaOH
aqueous solution) as reference standard.23
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures S1−S8, experimental details, and synthesis procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
ihamachi@sbchem.kyoto-u.ac.jp
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Prof. Teruyuki Nagamune (The University of Tokyo) and Dr.
Shinya Tsukiji (Nagaoka University of Technology) are
thanked for the plasmid encoding pBAD-DHFR. Prof. K.
Matsuda (Kyoto University) and Dr. M. Ikeda (Kyoto
University, Hamachi Lab) are acknowledged for their help
with DLS and AFM measurements, respectively. K.M. acknowl-
edges JSPS Research Fellowships for Young Scientists. This
work was partly supported by the CK Integrated Medical
Bioimaging Project (MEXT) and CREST (Japan Science and
Technology Agency).
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