Multicolor Activatable Raman Probes for Simultaneous Detection of Plural Enzyme Activities

Article abstract of DOI:10.1021/jacs.0c09200

Raman probes based on alkyne or nitrile tags hold promise for highly multiplexed imaging. However, sensing of enzyme activities with Raman probes is difficult because few mechanisms are available to modulate the vibrational response. Here we present a gen

Full text of DOI:10.1021/jacs.0c09200

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Multicolor Activatable Raman Probes for Simultaneous Detection of
Plural Enzyme Activities
Hiroyoshi Fujioka, Jingwen Shou, Ryosuke Kojima, Yasuteru Urano, Yasuyuki Ozeki,*
and Mako Kamiya*
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ABSTRACT: Raman probes based on alkyne or nitrile tags hold promise for
highly multiplexed imaging. However, sensing of enzyme activities with Raman
probes is difficult because few mechanisms are available to modulate the
vibrational response. Here we present a general strategy to prepare activatable
Raman probes that show enhanced Raman signals due to electronic
preresonance (EPR) upon reaction with enzymes under physiological
conditions. We identified a xanthene derivative bearing a nitrile group at
position 9 (9CN-JCP) as a suitable scaffold dye, and synthesized four types of
activatable Raman probes, which are targeted to different enzymes (three
aminopeptidases and a glycosidase) and tuned to different vibrational
frequencies by isotope editing of the nitrile group. We validated the activation
of the Raman signals of these probes by the target enzymes and succeeded in
simultaneous imaging of the four enzyme activities in live cells. Different cell
lines showed different patterns of these enzyme activities.
∼103 times, by positioning the pump wavelength at 100−200
nm longer wavelength than the molecular absorption
maximum. The EPR-SRS strategy provides submicromolar
sensitivity, together with spatial resolution comparable to that
of typical confocal fluorescence microscopy, and thus is
suitable for observing low-abundance molecules in cells.
However, most pre-existing Raman probes show constant
Raman signal intensity (“always-on” Raman probes), and thus
their application has been limited to labeling tags. Although
there are a few reports of Raman probes that show a shift in
Raman frequency (in response to a change of pH,14 or the
presence of hydrogen sulfide15) or that show Raman signal
activation by chemically producing alkynes in a lipid bilayer via
external addition of reagents,16 it has generally been believed
that the Raman signal cannot be switched on or off, because it
arises from molecular vibration. Therefore, “activatable-type”
Raman probes that can react with endogenous biological
targets and show an increased Raman signal under
physiological conditions have not been developed.
INTRODUCTION
■
Raman microscopy, which can characterize molecular species
by detecting the vibrations of chemical bonds, has been
developed as an important imaging modality to characterize
cellular biochemical constituents without labeling.1−3 Although
the imaging speed of Raman microscopy has been limited by
the weak signal intensity of spontaneous Raman scattering, the
emergence and evolution of stimulated Raman scattering
(SRS) microscopy, which exploits two-color laser pulses (the
pump beam and the Stokes beam), has improved the
sensitivity by several orders of magnitude, enabling high-
speed Raman imaging.4−6 Moreover, in the past decade,
various Raman tags, including alkyne, nitrile and the carbon−
deuterium bond, which show characteristic vibrational
frequencies in the cell-silent region (1800−2800 cm−1), have
been developed for bioorthogonal imaging of small-molecular
metabolites in live cells and tissues.7−9
Further, compared to fluorescence imaging, which suffers
from a “color barrier” (i.e., the number of simultaneously
resolvable targets is limited to four to six) owing to the
intrinsically broad fluorescence peaks, Raman imaging has
superior potential for highly multiplexed detection10 because
the Raman spectral line width can be 50−200 times smaller
than the fluorescence peak width. Min et al. have achieved
supermultiplexed vibrational imaging with two kinds of Raman
probes, called MARS11 and Carbow.12 In addition, SRS
imaging with MARS dyes can utilize the electronic
preresonance (EPR) effect13 to enhance the Raman signal by
Here we set out to establish a first-in-class general design
strategy of multicolor activatable Raman probes for simulta-
Received: August 27, 2020
https://dx.doi.org/10.1021/jacs.0c09200
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© XXXX American Chemical Society
A

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Figure 1. 9CN-pyronines as scaffold dyes of activatable Raman probes. (a) Chemical structures of 9CN-pyronines (see Table 1 for detailed
chemical structures and compound names). (b) Normalized absorption spectra of 9CN-pyronines measured in PBS (pH 7.4) containing 0.1%
DMSO as a cosolvent. (c) Normalized SRS spectra of 1 mM 9CN-pyronines measured in DMSO. (d) Time-dependent changes in the absorbance
of 9CN-pyronines measured in 200 mM sodium phosphate buffer (pH 7.4) containing 0.1% DMSO as a cosolvent. (e) Dose−response curves of
9CN-pyronines versus GSH concentration (0−25 mM). Absorbance was normalized at the wavelength of the respective absorption maxima shown
in Table 1. (f) Chemical structures of Ac-9CN-JCP and 9CN-JCP. The pictures showed 10 μM solutions of Ac-9CN-JCP and 9CN-JCP in PBS
(pH 7.4). (g) Absorption spectra of 1 μM Ac-9CN-JCP and 9CN-JCP measured in PBS (pH 7.4) containing 0.1% DMSO as a cosolvent. (h) SRS
spectra of 1 mM Ac-9CN-JCP and 9CN-JCP measured in PBS (pH 7.4) containing 10% DMSO as a cosolvent.
neous detection of plural biomolecules. For this purpose, we
focused on the phenomenon that the Raman signal intensity is
remarkably enhanced under EPR conditions compared to that
under electronic nonresonance conditions. Considering that
the wavelength of the pump beam in a typical SRS system is
around 800−900 nm, we speculated that the Raman signal
intensity could be “switched on”, i.e., highly activated, when
the molecular absorption of dyes exhibiting a Raman signal is
shifted from the visible (electronic nonresonance condition) to
the near-infrared (NIR) region (EPR condition) upon reaction
with the target biomolecules. To develop activatable Raman
probes based on this molecular design, we focused on the
activities of hydrolases in living cells, because it is well-known
that specific enzyme-catalyzed hydrolysis reactions can induce
significant absorption shifts of colorimetric/fluorogenic sub-
strates.17 We synthesized a series of Raman-active xanthene
derivatives, and among them we identified a suitable scaffold
for developing activatable Raman probes to target enzyme
activities. On the basis of this scaffold, we then prepared four
activatable Raman probes with distinct Raman peaks, which
exhibit Raman signal activation upon reaction with selected
enzymes of interest both in vitro and in living cells, and
succeeded in the simultaneous detection of plural enzyme
activities.
RESULTS AND DISCUSSION
■
Design of a Scaffold Dye for EPR-SRS Imaging in Live
Cells. To develop activatable Raman imaging probes that show
a red shift in absorption from the visible (electronic
nonresonance condition) to the NIR region (EPR condition)
upon reaction with enzymes, we first searched for a suitable
scaffold dye. For this purpose, we focused on xanthene
derivatives with the nitrile at the 9th position (9CN-
pyronines), because they display a single, sharp, and strong
Raman peak in the cell-silent region (1800−2800 cm−1) under
EPR conditions.11 To switch the Raman signal intensity on and
off, we prepared a series of symmetric and asymmetric 9CN-
pyronines bearing a primary aromatic amine at the C3 position
that would be available for conjugating enzyme substrate
moieties via an amide bond to blue-shift the molecular
B
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absorption18 (Figure 1a, Table 1, Schemes S1−S6). Consid-
measurement. We examined the dependence of the absorbance
of these 9CN-pyronines on GSH concentration (Figure 1e).
Most of the 9CN-pyronines showed a significant decrease of
absorbance in the presence of millimolar concentrations of
GSH. Among them, only 9CN-JCP exhibited sufficiently high
absorbance, suggesting that it would be stable under
physiological conditions. Thus, we selected 9CN-JCP as a
candidate scaffold dye and prepared its acetylated derivative,
Ac-9CN-JCP, as a model enzyme substrate (Figure 1f, Scheme
S7).
ering that the wavelength of the pump beam in our SRS
Table 1. Photochemical Properties of 9CN-Pyronines
Next, we compared the absorption spectra of Ac-9CN-JCP
and 9CN-JCP in phosphate-buffered saline at pH 7.4 (PBS)
(Figure 1g). As expected, Ac-9CN-JCP exhibited a blue-shifted
absorption spectrum (absorption maximum at 506 nm in
PBS); thus, a hypsochromic shift of more than 100 nm was
induced by the acetylation of 9CN-JCP. The blue-shifted
absorption of Ac-9CN-JCP is within the electronic non-
resonance region, while that of 9CN-JCP is within the EPR
region. In principle, such a molecular absorption shift should
induce a large change of Raman signals. To confirm that this is
the case, we next measured the SRS spectra of 1 mM Ac-9CN-
JCP and 1 mM 9CN-JCP in PBS. Indeed, a remarkable change
of SRS signal intensity was observed; Ac-9CN-JCP showed no
peak, while 9CN-JCP showed a clear single peak (Figure 1h).
Moreover, the RIE (relative Raman intensity vs EdU)24 value
of 9CN-JCP under EPR conditions was calculated to be about
87, which is much higher than those of alkynes and diynes
under nonresonance conditions, whereas Ac-9CN-JCP under
nonresonance conditions was calculated to be about 1.5
(Figure S4). These results support the idea that the Raman
signal can be activated by controlling the electronic resonance
via molecular absorption shift and indicate that 9CN-JCP is a
promising scaffold for developing activatable Raman probes to
observe enzyme activities.
Development of Multicolor Activatable Raman
Probes for Plural Enzyme Activities. On the basis of the
newly developed 9CN-JCP scaffold, we next isotopically edited
the atoms of nitrile group on 9CN-JCPs to prepare 9C15N-
JCP, 913CN-JCP, and 913C15N-JCP, to shift the vibrational
resonances8 (Figure 2a, Scheme S8). As expected, these
isotope-edited 9CN-JCPs exhibited single sharp Raman peaks
at distinct wavenumbers (Figure 2b). We further examined
whether these SRS signals can be identified as four separate
peaks even when the compounds are mixed. As expected, the
SRS spectrum of the solution mixture showed four separate
peaks, even though the absorption and fluorescence spectra of
the compounds were identical (Figure 2c, Figure S5).
Encouraged by these promising results, we next prepared
four activatable Raman probes targeted to different enzymes by
replacing the acetyl group of Ac-9CN-JCP with appropriate
enzyme substrate moieties. We incorporated a γ-L-glutamyl
(gGlu), L-leucyl (Leu), L-glutamyl-L-prolyl (EP), or β-D-
galactosyl (βGal) group into the isotope-edited 9CN-JCPs
via an amide bond or a carbamate linker to target γ-glutamyl
transpeptidase (GGT), leucine aminopeptidase (LAP),
dipeptidyl peptidase-4 (DPP-4), and β-galactosidase (β-Gal),
respectively (Figure 2a, Schemes S9−S12). We confirmed that
these probes (gGlu-9CN-JCP, Leu-9C15N-JCP, EP-913CN-
JCP, βGal-913C15N-JCP) exhibited relatively short-wavelength
absorption similar to that of Ac-9CN-JCP but were converted
to the respective scaffolds (9CN-JCP, 9C15N-JCP, 913CN-JCP,
913C15N-JCP) upon reaction with corresponding target
enzymes, resulting in a significant red shift in absorption
λabs
ωR
a
b
compound
X
R1
R2
R3
[nm]
[cm−1
]
9CN-SiP
9CN-CP
9CN-diMeSiP
9CN-diMeCP
9CN-JSiP
SiMe2
CMe2
SiMe2
CMe2
SiMe2 −(CH2)3−
CMe2
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
673
646
708
680
650
630
506
2217
2227
2216
2223
2214
2220
9CN-JCP
Ac-9CN-JCP
−(CH2)3−
−(CH2)3− CH3CO
c
CMe2
n.d.
a
Measured in PBS (pH 7.4) containing 0.1% DMSO as a cosolvent.
Measured in DMSO. n.d. means not detected under 1 mM
condition. λabs: Absorption maximum. ωR: Raman wavenumber of
CN vibrational mode.
b
c
imaging system is fixed at 843 nm to acquire Raman signals in
the 2000−2300 cm−1 region (Figure S1),19 the wavelength
region of molecular absorption appropriate for EPR is expected
to be 620−750 nm (see Supporting Information).11 As it has
been reported that 9CN-xanthenes with oxygen (O) at the
10th position tend to exhibit relatively short wavelengths,20,21
we focused on silicon (Si) or carbon (C) as the atom at the
10th position of xanthene (X in Figure 1a). For N-substitution
at C6, we selected a primary amine (for 9CN-SiP, 9CN-CP),
an N,N-dimethylamine (for 9CN-diMeSiP, 9CN-diMeCP), or
a julolidine-like structure (for 9CN-JSiP, 9CN-JCP). All of the
synthesized compounds exhibited suitable absorption spectra
that overlapped well with the EPR region and showed
sufficiently strong SRS signals in the silent region under EPR
conditions (Figure 1b,c, Table 1). Among these derivatives,
9CN-JSiP and 9CN-JCP, which have julolidine-like structure,
exhibited distinct spectra in terms of both peak positions and
band widths; this may be due to the environmental sensitivity
of these derivatives, because they showed red-shifted
absorption spectra in DMSO (Figure S2). Next, we examined
the stability of these 9CN-pyronines under physiological
conditions by evaluating their stability in phosphate buffer at
pH 7.4 and their resistance to glutathione (GSH), one of the
major antioxidants in cells. Because some pyronines are
attacked at the 9th carbon atom of the xanthene ring by
intracellular nucleophiles such as H2O or GSH,22 and these
reactions convert the dyes to a colorless form, we thought this
would lead to an electronic nonresonance state with a reduced
Raman response. When we examined the stability in phosphate
buffer at pH 7.4, the absorbance of 9CN-SiP and 9CN-CP
significantly decreased after 2 h incubation at room temper-
ature, with formation of the respective xanthones, due to the
nucleophilic attack of H2O (Figure 1d, Figure S3). As regards
resistance to GSH, the intracellular GSH concentration is
reported to be in the sub-millimolar to a few millimolar
range,23 so we expected that scaffold dyes which show constant
NIR absorbance even in the presence of millimolar
concentrations of GSH would be favorable for stable
C
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Figure 2. Development of activatable Raman probes for enzyme activities based on isotope-edited 9CN-JCPs. (a) Molecular design of activatable
Raman probes based on isotope-edited 9CN-JCPs (9CN-JCP (red), 9C15N-JCP (green), 913CN-JCP (blue), 913C15N-JCP (yellow)). Raman
signals from 9CN-JCP-based probes are activated upon reaction of the probe with the target enzyme because the absorption is shifted from the
visible (electronic nonresonance conditions) to the NIR region (EPR conditions). gGlu-9CN-JCP for γ-glutamyl transpeptidase (GGT), Leu-
9C15N-JCP for leucine aminopeptidase (LAP), EP-913CN-JCP for dipeptidyl peptidase-4 (DPP-4), βGal-913C15N-JCP for β-galactosidase (β-Gal).
(b) Normalized SRS spectra of 1 mM isotope-edited 9CN-JCPs measured in DMSO. The spectra are vertically offset for clarity. (c) SRS spectrum
of a mixture of the isotope-edited 9CN-JCPs (250 μM each) measured in DMSO. Highlights represent the regions of vibrational frequency of
9CN-JCP (red), 9C15N-JCP (green), 913CN-JCP (blue), and 913C15N-JCP (yellow). (d) SRS spectra of 1 mM gGlu-9CN-JCP, Leu-9C15N-JCP,
EP-913CN-JCP or βGal-913C15N-JCP before (gray) and after (black) reaction with 1 unit of GGT, 0.12 units of LAP, 0.022 units of DPP-4, or 1
unit of β-Gal measured in PBS (pH 7.4) containing 10% DMSO as a cosolvent. Reaction solutions were incubated for 1 h at room temperature. (e)
SRS spectra of a mixture of the isotope-edited 9CN-JCP probes (250 μM each) before (gray) and after (black) reaction with 5 units of GGT and
10 units of β-Gal (left) or with 0.12 units of LAP and 0.044 units of DPP-4 (right) measured in PBS (pH 7.4) containing 10% DMSO as a
cosolvent. Reaction solutions were incubated for 1 h at room temperature.
(Figures S6−S9). We also confirmed that the Raman signal of
each probe was significantly activated upon enzyme reaction,
We next examined whether the four different enzyme
activities can be evaluated simultaneously by using a mixture of
9CN-JCP probes (gGlu-9CN-JCP, Leu-9C15N-JCP, EP-
913CN-JCP, βGal-913C15N-JCP). Indeed, we observed specific
SRS signal activation by the corresponding enzyme activities.
Specifically, SRS signals of 9CN-JCP (2217 cm−1) and
913C15N-JCP (2137 cm−1) were detected in the presence of
GGT and β-Gal, and SRS signals of 9C15N-JCP (2190 cm−1)
and 913CN-JCP (2166 cm−1) were detected in the presence of
LAP and DPP-4 (Figure 2e). These results indicate that
isotope-edited 9CN-JCP-based activatable Raman probes can
simultaneously detect plural enzyme activities.
and the vibrational frequencies (2217 cm−1 2190 cm−1, 2166
,
cm−1, 2137 cm−1) corresponded to the vibrational frequencies
of the triple bond of 9CN-JCP, 9C15N-JCP, 913CN-JCP, and
913C15N-JCP in PBS (Figure 2d). We also confirmed that the
activation of these probes was inhibited by coincubation with
inhibitors of the corresponding enzymes (Figures S6−S9), and
the Raman signal intensity produced by gGlu-9CN-JCP was
linearly correlated with the concentration of GGT, demon-
strating the suitability of this system for quantitative detection
(Figure S10). To our knowledge, this is the first demonstration
of activatable Raman probes that can report the activities of
various hydrolases under physiological conditions.
Multicolor Imaging of Target Enzyme Activities in
Live Cells. Next, we examined the applicability of our isotope-
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Figure 3. Simultaneous detection of the four enzyme activities in live cells by SRS imaging. A549 cells (left) and H226 cells (right) cells were
incubated with 10 μM gGlu-9CN-JCP, Leu-9C15N-JCP, and EP-913CN-JCP, and 20 μM βGal-913C15N-JCP in HBSS (+) containing 0.5% DMSO
as a cosolvent for 30 min. (a) SRS images of the four enzyme activities in A549 cells (left) and H226 cells (right). Images were obtained at 2217
cm−1 for gGlu-9CN-JCP, 2190 cm−1 for Leu-9C15N-JCP, 2166 cm−1 for EP-913CN-JCP, and 2137 cm−1 for βGal-913C15N-JCP. The background
image at 2250 cm−1 was subtracted in each case. Scale bar: 10 μm. The image acquisition time was 167 s. (b) SRS spectra of enzyme activities
obtained from A549 cells (left) and H226 cells (right). Spectra were obtained from the ROIs shown in Figure S23.
edited 9CN-JCP probes for the detection of enzyme activities
in live cells. For this purpose, we selected two human lung
carcinoma cell lines A549 and H226, which we found to have
different patterns of enzyme activities by using our previously
reported HMRG-based fluorescence probes for GGT,25 LAP,26
and DPP-427 and our HMRef-based fluorescence probe for β-
Gal,28 and by measuring the gene expression levels with
quantitative real-time PCR. These results showed that the
activities and expression levels of GGT and β-Gal are higher in
A549 cells, while those of LAP and DPP-4 are higher in H226
cells (Figures S11 and S12).
We first applied our 9CN-JCP probes individually to live
A549 cells and H226 cells to examine whether the probes
could visualize the enzyme activities in cells under our SRS
microscope. After 30 min incubation with 10 μM gGlu-9CN-
JCP, Leu-9C15N-JCP, or EP-913CN-JCP or with 20 μM βGal-
913C15N-JCP, specific SRS signal activations were observed
(Figures S13−S16). Moreover, by taking advantage of the faint
red fluorescence emission from the 9CN-JCP scaffolds (Φfl =
0.055 in PBS(−)), we confirmed that the fluorescence signals
of the activated 9CN-JCP probes colocalized well with the SRS
signals by dual-mode imaging of SRS and fluorescence (Figures
S13−S16). Further, the subcellular localization of these SRS/
fluorescence signals is colocalized well with that of
LysoTracker Green, demonstrating that the cleaved dyes
(9CN-JCPs) tend to accumulate mainly in lysosomes (Figure
S17). We also confirmed that the activation of each probe was
inhibited by coincubation with an inhibitor of the correspond-
ing enzyme (Figures S18−S21). The obtained SRS signal
intensities were well correlated with the fluorescence
intensities, suggesting that evaluation of these enzyme activities
with our newly developed Raman probes is reliable (Figure
S22).
Finally, we applied our four 9CN-JCP probes simultaneously
to A549 cells or H226 cells to examine whether the probes can
visualize the different patterns of the four enzyme activities in
A549 cells and H226 cells. As shown in Figure 3a,b, the four
different enzyme activities were clearly detected as SRS images
at distinct Raman frequencies, and the SRS intensities were
consistent with those seen when the probes were applied
individually. Further, we observed a relatively high background
signal in the spectra in cellulo, which is likely to be derived
from two-photon absorption (TPA)29 in EPR condition and
cross-phase modulation (XPM),5,29 which tend to become
significant in the presence of cells. However, this background
would not affect the accuracy of detection, because the sharp
signals of the probes are superimposed on the background,
which can easily be subtracted if required. Because these 9CN-
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JCP probes are indistinguishable by fluorescence microscopy,
this simultaneous detection of the four enzyme activities is a
consequence of the sharp Raman signatures (fwhm of the
nitrile peak of our 9CN-JCPs is about 15 cm−1), which are
highly advantageous for multiplexed imaging. It is particularly
noteworthy that the enzyme activities could be detected by
using probe concentrations as low as 10−20 μM, owing to the
very strong signal activation by shifting molecular absorption
from electronic nonresonance conditions to EPR-SRS
conditions, and thus the sensitivity is quite high as Raman
imaging. Moreover, the spatial resolution of SRS microscopy
has been reported to be around 300−400 nm,10 which is
comparable to that of typical confocal fluorescence micros-
copy. Further, the acquisition time for obtaining SRS images at
five different wavenumbers (four wavenumbers for enzyme
activities and one wavenumber for background subtraction)
was only 2−3 min with our SRS imaging system, which is
suitable for live imaging of biomolecules. As we employed
1000-fold signal averaging in these experiments, it would be
possible to shorten the acquisition time by averaging fewer
signals.
alized Raman probes will be a powerful tool for studying
complex biological and pathological phenomena.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c09200.
Materials and methods, synthetic procedures and
characterization of compounds, detailed experimental
procedures, spectroscopic data, fluorescence, and SRS
imaging results (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Yasuyuki Ozeki − Department of Electrical Engineering and
Information Systems, Graduate School of Engineering, The
University of Tokyo, Tokyo 113-0033, Japan; Email: ozeki@
ee.t.u-tokyo.ac.jp
Mako Kamiya − Graduate School of Medicine, The University
of Tokyo, Tokyo 113-0033, Japan; orcid.org/0000-0002-
5592-1849; Email: mkamiya@m.u-tokyo.ac.jp
CONCLUSION
■
In summary, we have established a first-in-class general strategy
to prepare activatable Raman probes that show significant
enhancement of Raman signals due to a red shift of the
molecular absorption upon reaction with the target enzymes.
We synthesized several candidate 9CN-pyronines, and
evaluation of their optical properties and stability under
physiological conditions identified 9CN-JCP as a promising
scaffold for activatable Raman probes. We then developed four
kinds of activatable Raman imaging probes, gGlu-9CN-JCP,
Leu-9C15N-JCP, EP-913CN-JCP, and βGal-913C15N-JCP,
which exhibit separate sharp Raman peaks and are targeted
to different enzyme activities. By applying these four
activatable probes to live cells, we succeeded in simultaneous
imaging of the four enzyme activities in live cells. To our
knowledge, this is the first demonstration of activatable Raman
probes that can report enzyme activities under physiological
conditions. To expand the color palette, we are planning to
develop other scaffolds with peak numbers 15 cm−1 apart from
that of 9CN-JCP, which might enable us to add four more
colors, considering that the fwhm of the nitrile peak of 9CN-
JCPs is about 15 cm−1 and that isotope-editing shifts the peak
wavenumber by about 30 cm−1. To further expand the color
variation, we are also planning to replace the nitrile group with
an alkyne moiety, which has a peak wavenumber well away
from those of nitriles (at around 2180 cm−1). We also
anticipate that the wavenumber of the alkyne group can be
varied more markedly by the chemical modification, thus
opening up the prospect of supermultiplex imaging. Because
various enzyme activities are up- or down-regulated in many
diseases,30,31 methodology for simultaneous detection of plural
enzyme activities is expected to increase our understanding of
the heterogeneity and complexity of diseases. These probes are
also promising candidates as tools for precise and early
diagnosis of disease. Furthermore, our design strategy for
activatable Raman probes should be generally applicable for
developing activatable Raman probes targeted to other specific
biomolecules or biological processes by leveraging available
molecular design strategies for activatable “fluorescent” probes.
Therefore, we believe multiplex imaging with these function-
Authors
Hiroyoshi Fujioka − Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo 113-0033, Japan;
orcid.org/0000-0003-1904-0566
Jingwen Shou − Department of Electrical Engineering and
Information Systems, Graduate School of Engineering, The
University of Tokyo, Tokyo 113-0033, Japan
Ryosuke Kojima − Graduate School of Medicine, The
University of Tokyo, Tokyo 113-0033, Japan; PRESTO,
Japan Science and Technology Agency, Saitama 332-0012,
Japan; orcid.org/0000-0002-3792-9222
Yasuteru Urano − Graduate School of Pharmaceutical
Sciences and Graduate School of Medicine, The University of
Tokyo, Tokyo 113-0033, Japan; orcid.org/0000-0002-
1220-6327
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c09200
Author Contributions
All authors contributed to the writing of the manuscript and
have given approval to the final version of the manuscript.
Notes
The authors declare the following competing financial
interest(s): H.F., J.S., Y.U., Y.O., and M.K are inventors of a
patent application covering activatable Raman probes.
ACKNOWLEDGMENTS
■
This research was supported in part by MEXT/JSPS
KAKENHI grants JP15H05951 “Resonance Bio”,
JP19H02826, and JP19K22242, JP20H05723, JP20H05724
(to M.K.), JSPS KAKENHI JP20H02650, JP20H05725 (to
Y.O.), JP19J22546 (to J.S.), by JSPS Core-to-Core Program
(JPJSCCA20170007), A. Advanced Research Networks, by
JST CREST JPMJCR1872 (to Y.O.), by Japan Foundation for
Applied Enzymology (to M.K.), by The Naito Foundation (to
M.K.), and by Quantum Leap Flagship Program of MEXT
JPMXS0118067246.
F
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Article
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