A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria

Article abstract of DOI:10.1016/j.bmcl.2014.11.080

We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits an intense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Raman probe by linking bisphenylbutadiyne with triphenylphosphonium, a well-known mitochondrial targeting moiety. This probe, named MitoBADY, has a Raman peak 27 times more intense than that of 5-ethynyl-2′-deoxyuridine. Raman microscopy using submicromolar extracellular probe concentrations successfully visualized mitochondria in living cells. A full Raman spectrum is acquired at each pixel of the scanned sample, and we showed that simultaneous Raman imaging of MitoBADY and endogenous cellular biomolecules can be achieved in a single scan. MitoBADY should be useful for the study of mitochondrial dynamics.

Full text of DOI:10.1016/j.bmcl.2014.11.080

Bioorganic & Medicinal Chemistry Letters 25 (2015) 664–667
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A sensitive and specific Raman probe based on bisarylbutadiyne for
live cell imaging of mitochondria
Hiroyuki Yamakoshi a,b, Almar Palonpon , Kosuke Dodo a,b,c, Jun Ando a,b,c,d, Satoshi Kawata d,
a,d
a,c,d
a,b,c,
⇑
Katsumasa Fujita
, Mikiko Sodeoka
a
Sodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technology Agency, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
CREST, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b
c
d
Department of Applied Physics, Osaka University, 2-1 Yamadaoka Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits an
intense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Raman
probe by linking bisphenylbutadiyne with triphenylphosphonium, a well-known mitochondrial targeting
Received 24 October 2014
Revised 26 November 2014
Accepted 28 November 2014
Available online 5 December 2014
0
moiety. This probe, named MitoBADY, has a Raman peak 27 times more intense than that of 5-ethynyl-2 -
deoxyuridine. Raman microscopy using submicromolar extracellular probe concentrations successfully
visualized mitochondria in living cells. A full Raman spectrum is acquired at each pixel of the scanned
sample, and we showed that simultaneous Raman imaging of MitoBADY and endogenous cellular biomol-
ecules can be achieved in a single scan. MitoBADY should be useful for the study of mitochondrial
dynamics.
Keywords:
Raman
Imaging
Mitochondria
Triphenylphosphonium
Live cell
Ó 2014 Elsevier Ltd. All rights reserved.
Mitochondria play a central role in cell maintenance, including
energy supply, cell death, regulation of calcium signaling and
cellular metabolism. They have been the target of a number of bio-
active molecules, drug candidates and functionalized molecules
c, an endogenous protein abundant in the mitochondrial mem-
brane. However, the distribution and concentration of cytochrome
4
c vary with cell types and conditions, and therefore cytochrome c is
not always a reliable probe for mitochondria. For instance, during
apoptosis, cytochrome c is released into the cytosol from mitochon-
1
designed to induce various biological effects. The ability to image
5
mitochondria in live cells is extremely useful for understanding
mitochondrial dynamics and its association with various biological
processes. Fluorescence methods employing mitochondrion-stain-
ing probes are well suited for this purpose because of their high
sensitivity and specificity.2
dria. In addition, the Raman intensity of cytochrome c peaks is
dependent upon the redox state, which in turn depends strongly
6
on the physiological state of the cell. Other types of cytochromes,
such as cytochrome b, which exhibits similar Raman peaks, might
7
also contribute to the detected Raman signals. Other intrinsic
On the other hand, Raman microscopy is available as a comple-
mentary imaging technique that does not require any label, but
uses scattered light from vibrations of biomolecules. Spatially
resolved Raman spectra obtained by Raman microscopy reflect
the local biochemical composition of the cell, and thus offer more
information than is available from fluorescence probes. Label-free
imaging of mitochondrial distribution in cells has been demon-
strated using confocal Raman microscopy combined with multivar-
iate image construction methods.3 Mitochondria have also been
visualized in cells in a simpler way, without the need for multivar-
iate analysis, by detecting the resonant Raman peaks of cytochrome
Raman signals from the cell could possibly be used, but this is
problematic because of poor molecular selectivity owing to the
marked overlap of Raman signals from a variety of endogenous
molecules.
Instead of using intrinsic Raman signals to obtain imaging con-
trast, an alternative strategy is to use an exogenous Raman probe
whose signal appears in the Raman-silent region of the cell
ꢀ1
(1800–2800 cm ). Efforts along this direction have led us to
develop alkyne-tag Raman imaging (ATRI) for visualization of spe-
8
cific small molecules in live cells. In this technique, we tag mole-
cules with an alkyne moiety whose vibrational frequency lies in
the silent region of the cell, free from the interfering signals of
endogenous molecules, and which is small enough to minimally
9
perturb the behavior of the target small molecule. We also inves-
⇑
Corresponding author. Tel.: +81 48 467 9373; fax: +81 48 462 4666.
E-mail address: sodeoka@riken.jp (M. Sodeoka).
tigated the structure-Raman shift/intensity relationships of alkynes
http://dx.doi.org/10.1016/j.bmcl.2014.11.080
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
0

H. Yamakoshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 664–667
665
and demonstrated that simultaneous imaging of two small
molecules having different Raman shifts was possible. Among the
different alkynyl structures that we tested, bisarylbutadiynes
(
BADY; Fig. 1a) showed the strongest Raman intensity, producing
0
alkyne peaks about 25 times stronger than that of 5-ethynyl-2 -
deoxyuridine (EdU) on average.8b Although BADY is a bulky Raman
tag for visualization of small molecules, we expected that it could
be used as a sensitive and specific Raman probe for visualizing
cellular organelles such as mitochondria after addition of an
appropriate targeting moiety. Here, we describe the synthesis of a
mitochondrion-targeting Raman probe based on BADY (MitoBADY;
Fig. 1b) and we demonstrate its utility for imaging mitochondria in
live cells.
Scheme 1. Synthesis of MitoBADY. Reagents and conditions: (a) Ethynylbenzene,
CuI, NiCl
2
ꢁ6H
2
O, tetramethylethylenediamine, air, THF; (b) LiAlH
4
, THF, 0 °C, 52% for
As a mitochondrial targeting moiety, we selected triphenylphos-
phonium, a liphophilic cation, that is accumulated in the negatively
2
steps; (c) MsCl, Et
3
N, CH Cl , 76%, (d) PPh , NaI, MeCN, 75 °C, 95%.
2
2
3
10
charged mitochondrial matrix. It has been widely used to target
1
1
12
anti-oxidants,
fluorogenic sensors,
photoactivated delivery
1
3
14
systems and other molecules to mitochondria.
MitoBADY was easily synthesized from commercially available
4
-ethynylbenzaldehyde (Scheme 1). The bisarylbutadiyne structure
1
5
was constructed by means of Ni-catalyzed yne–yne coupling.
After reduction of the aldehyde, the benzylic alcohol was converted
to triphenylphosphonium in two steps. In the Raman spectrum of
ꢀ1
the synthesized MitoBADY, the alkyne peak at 2218 cm showed
a relative Raman intensity vs EdU (RIE) of 27, in agreement with
8
b
our previous report. The negligible fluorescence background in
the Raman spectrum and the observed UV–vis absorption spectrum
confirmed that MitoBADY is nonfluorescent upon visible light
1
6
excitation.
To investigate the potential of MitoBADY for live-cell imaging of
mitochondria, HeLa cells were treated with 400 nM MitoBADY, and
Raman images were acquired using a home-built slit-scanning
1
7
Raman microscope.
At this concentration of MitoBADY, no
precipitation was observed during the cellular experiments. Fig-
ure 2a shows the distributions of cytochrome c and MitoBADY,
ꢀ1
ꢀ1
observed at 751 cm and 2220 cm , respectively, at various incu-
bation times. The contrast of MitoBADY was observable within
5
min, indicating rapid uptake by the cells, and reached a steady
1
8
state in 30 min, remaining relatively stable for at least 75 min.
The MitoBADY distribution coincided well with that of cytochrome
c distribution in the colocalization images. The time-dependent
uptake of MitoBADY was further supported by the relative intensi-
ties of the cytochrome c and MitoBADY peaks in the Raman spectra
obtained from the cytosol (Fig. 2b). Since the cells in this experi-
ment are non-apoptotic, the distribution of mitochondria is
expected to be the same as that of cytochrome c, as was verified
in subsequent experiments (see Fig. 3). Thus, our results show that
MitoBADY selectively targets and accumulates in mitochondria.
The mitochondrial localization of MitoBADY in live cells was
further confirmed by comparing the distribution of MitoBADY with
that of a typical mitochondrion probe, MitoTracker Green FM (MTG).
Live HeLa cells treated with and without 200 nM MitoBADY were
stained with MTG for 10 min. The cells were then subjected to suc-
cessive fluorescence and Raman imaging using the same slit-scan-
ning microscope setup. These images coincided well, confirming
(
a)
(b)
I
Ph
Ph
P
Ph
Ar'
Figure 2. Raman imaging of MitoBADY in living cells. (a) Raman images showing
ꢀ
1
ꢀ1
the distributions of cytochrome c at 751 cm (red) and MitoBADY at 2220 cm
Ar
bisarylbutadiyne (BADY)
Figure 1. Structures of bisarylbutadiyne (BADY) and MitoBADY.
(green) in live HeLa cells treated with 400 nM MitoBADY. Areas of colocalization in
the merged image appear yellow. (b) Average Raman spectra obtained from the
MitoBADY (1)
cytosol showing relative intensities of cytochrome c and MitoBADY peaks. Imaging
2
conditions: laser wavelength, 532 nm; laser intensity, 3.3 mW/
5 s/line. Scale bar is 10 m.
l
m ; exposure time,
l

6
66
H. Yamakoshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 664–667
Figure 3. Sequential imaging of MTG and MitoBADY in living cells. Fluorescence and Raman images of HeLa cells treated with (a) 50 nM MTG and 200 nM MitoBADY and (b)
2
5
0 nM MTG only. The fluorescence imaging was done first, followed by Raman imaging. Imaging conditions: laser wavelength, 532 nm; laser intensity, 0.05 mW/lm
2
(
fluorescence), 3.3 mW/lm (Raman); exposure time, 0.2 s/line (fluorescence), 5 s/line (Raman); Scale bar is 10 lm.
the selective staining capability of MitoBADY for mitochondria
Fig. 3). In addition, the Raman image of cytochrome c shows the
(
same distribution as that of MTG, establishing cytochrome c as
another good Raman probe for mitochondria in the case of
non-apoptotic cells under resonance excitation conditions. Note
that the high laser intensity used in Raman imaging causes photo-
bleaching of the MTG fluorophores so that no fluorescence from
MTG is detected during Raman imaging; otherwise, this might
interfere with the measurement of the Raman signals.
Since a full Raman spectrum is acquired at each pixel of the
scanned sample, simultaneous Raman imaging of MitoBADY and
endogenous cellular biomolecules is readily available from a
single scan. As shown in Figure 4, the subcellular pattern of
MitoBADY, which represents the distribution of mitochondria, is
quite distinct from the patterns of endogenous protein and lipid,
ꢀ
1
as identified from the amide-I (1686 cm ) and CH
(
2
stretching
ꢀ1
2853 cm ) vibrations, respectively. This result demonstrates that
organelle-specific Raman probes such as MitoBADY can be used to
image specific cellular targets simultaneously with intrinsic biomol-
ecules, thereby providing a great deal of chemical information about
the cell.
In summary, we have synthesized and characterized a novel
mitochondrion-targeting Raman probe called MitoBADY and
applied it to cellular imaging of mitochondria. The bisphenyl-substi-
tuted diyne structure provides a Raman signal intensity 27 times
higher than that of EdU, enabling application of the probe at
submicromolar extracellular concentrations. Since the signal of
MitoBADY appears in the Raman-silent region of the cell, it is possible
Figure 4. Simultaneous Raman imaging of MitoBADY and endogenous biomole-
cules in living cells. Raman images show the subcellular distributions of protein,
MitoBADY, and lipid in live HeLa cells treated with 200 nM MitoBADY. Color
channels in the merged image: blue, protein; green, MitoBADY; red, lipid. Imaging
2
conditions: laser wavelength, 532 nm; laser intensity, 2.3 mW/
5 s/line; Scale bar is 10 m.
l
m ; exposure time,
l

H. Yamakoshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 664–667
667
to obtain Raman signals simultaneously from other endogenous
compounds or exogenous molecules in the cell, making it possible
to visualize the relationship of cellular components to cellular
structures. It should be straightforward to develop Raman probes
targeting other organelles by using BADY coupled with other targeting
moieties, and this would extend the scope of Raman microscopy to
the molecular analysis of specific cellular domains.
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