Bioorganic & Medicinal Chemistry Letters 25 (2015) 664–667

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Bioorganic & Medicinal Chemistry Lettersjournal homepage: www.elsevier.com/locate/bmcl

A sensitive and speciﬁc Raman probe based on bisarylbutadiyne forlive cell imaging of mitochondria
Hiroyuki Yamakoshi a,b, Almar Palonpon a,d, Kosuke Dodo a,b,c, Jun Ando a,b,c,d, Satoshi Kawata d,
Katsumasa Fujita a,c,d, Mikiko Sodeoka a,b,c,⇑aSodeoka 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, JapanCREST, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japand
Department of Applied Physics, Osaka University, 2-1 Yamadaoka Suita, Osaka 565-0871, Japanbca r t i c l ei n f oArticle history:
Received 24 October 2014
Revised 26 November 2014
Accepted 28 November 2014
Available online 5 December 2014
Keywords:
RamanImaging
Mitochondria
TriphenylphosphoniumLive cella b s t r a c t
We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits anintense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Ramanprobe 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-20 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.
Ó 2014 Elsevier Ltd. All rights reserved.

Mitochondria play a central role in cell maintenance, including
energy supply, cell death, regulation of calcium signaling andcellular metabolism. They have been the target of a number of bioactive molecules, drug candidates and functionalized molecules
designed to induce various biological effects.1 The ability to image
mitochondria in live cells is extremely useful for understanding
mitochondrial dynamics and its association with various biologicalprocesses. Fluorescence methods employing mitochondrion-staining probes are well suited for this purpose because of their highsensitivity and speciﬁcity.2
On the other hand, Raman microscopy is available as a complementary imaging technique that does not require any label, butuses scattered light from vibrations of biomolecules. Spatiallyresolved 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 demonstrated using confocal Raman microscopy combined with multivariate image construction methods.3 Mitochondria have also beenvisualized in cells in a simpler way, without the need for multivariate analysis, by detecting the resonant Raman peaks of cytochrome⇑ Corresponding author. Tel.: +81 48 467 9373; fax: +81 48 462 4666.
E-mail address: sodeoka@riken.jp (M. Sodeoka).
http://dx.doi.org/10.1016/j.bmcl.2014.11.080
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

c, an endogenous protein abundant in the mitochondrial membrane.4 However, the distribution and concentration of cytochromec 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 mitochondria.5 In addition, the Raman intensity of cytochrome c peaks isdependent upon the redox state, which in turn depends stronglyon the physiological state of the cell.6 Other types of cytochromes,
such as cytochrome b, which exhibits similar Raman peaks, mightalso contribute to the detected Raman signals.7 Other intrinsic
Raman signals from the cell could possibly be used, but this isproblematic because of poor molecular selectivity owing to themarked overlap of Raman signals from a variety of endogenous
molecules.
Instead of using intrinsic Raman signals to obtain imaging contrast, an alternative strategy is to use an exogenous Raman probe
whose signal appears in the Raman-silent region of the cell(1800–2800 cm1). Efforts along this direction have led us todevelop alkyne-tag Raman imaging (ATRI) for visualization of speciﬁc small molecules in live cells.8 In this technique, we tag molecules with an alkyne moiety whose vibrational frequency lies inthe silent region of the cell, free from the interfering signals ofendogenous molecules, and which is small enough to minimally
perturb the behavior of the target small molecule.9 We also investigated the structure-Raman shift/intensity relationships of alkynesH. Yamakoshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 664–667

and demonstrated that simultaneous imaging of two small
molecules having different Raman shifts was possible. Among thedifferent alkynyl structures that we tested, bisarylbutadiynes(BADY; Fig. 1a) showed the strongest Raman intensity, producing
alkyne peaks about 25 times stronger than that of 5-ethynyl-20 deoxyuridine (EdU) on average.8b Although BADY is a bulky Ramantag 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 inlive cells.
As a mitochondrial targeting moiety, we selected triphenylphosphonium, a liphophilic cation, that is accumulated in the negatively
charged mitochondrial matrix.10 It has been widely used to target
anti-oxidants,11 ﬂuorogenic sensors,12 photoactivated delivery
systems13 and other molecules14 to mitochondria.
MitoBADY was easily synthesized from commercially available
4-ethynylbenzaldehyde (Scheme 1). The bisarylbutadiyne structure
was constructed by means of Ni-catalyzed yne–yne coupling.15
After reduction of the aldehyde, the benzylic alcohol was converted
to triphenylphosphonium in two steps. In the Raman spectrum of
the synthesized MitoBADY, the alkyne peak at 2218 cm1 showeda relative Raman intensity vs EdU (RIE) of 27, in agreement with
our previous report.8b The negligible ﬂuorescence background in
the Raman spectrum and the observed UV–vis absorption spectrum
conﬁrmed that MitoBADY is nonﬂuorescent upon visible lightexcitation.16
To investigate the potential of MitoBADY for live-cell imaging of
mitochondria, HeLa cells were treated with 400 nM MitoBADY, andRaman images were acquired using a home-built slit-scanning
Raman microscope.17 At this concentration of MitoBADY, noprecipitation was observed during the cellular experiments. Figure 2a shows the distributions of cytochrome c and MitoBADY,
observed at 751 cm1 and 2220 cm1, respectively, at various incubation times. The contrast of MitoBADY was observable within
5 min, indicating rapid uptake by the cells, and reached a steadystate in 30 min, remaining relatively stable for at least 75 min.18
The MitoBADY distribution coincided well with that of cytochromec distribution in the colocalization images. The time-dependent
uptake of MitoBADY was further supported by the relative intensities of the cytochrome c and MitoBADY peaks in the Raman spectra
obtained from the cytosol (Fig. 2b). Since the cells in this experiment are non-apoptotic, the distribution of mitochondria isexpected 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 wasfurther conﬁrmed by comparing the distribution of MitoBADY withthat 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 successive ﬂuorescence and Raman imaging using the same slit-scanning microscope setup. These images coincided well, conﬁrming

(a)

(b)

IAr'PhPPh
PhArbisarylbutadiyne (BADY)

MitoBADY (1 )Figure 1. Structures of bisarylbutadiyne (BADY) and MitoBADY.

665

Scheme 1. Synthesis of MitoBADY. Reagents and conditions: (a) Ethynylbenzene,CuI, NiCl26H2O, tetramethylethylenediamine, air, THF; (b) LiAlH4, THF, 0 °C, 52% for
2 steps; (c) MsCl, Et3N, CH2Cl2, 76%, (d) PPh3, NaI, MeCN, 75 °C, 95%.

Figure 2. Raman imaging of MitoBADY in living cells. (a) Raman images showing
the distributions of cytochrome c at 751 cm1 (red) and MitoBADY at 2220 cm1(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 thecytosol showing relative intensities of cytochrome c and MitoBADY peaks. Imaging
conditions: laser wavelength, 532 nm; laser intensity, 3.3 mW/lm2; exposure time,5 s/line. Scale bar is 10 lm.

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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)50 nM MTG only. The ﬂuorescence imaging was done ﬁrst, followed by Raman imaging. Imaging conditions: laser wavelength, 532 nm; laser intensity, 0.05 mW/lm2(ﬂuorescence), 3.3 mW/lm2 (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 thesame 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. Notethat the high laser intensity used in Raman imaging causes photobleaching of the MTG ﬂuorophores so that no ﬂuorescence from
MTG is detected during Raman imaging; otherwise, this mightinterfere with the measurement of the Raman signals.
Since a full Raman spectrum is acquired at each pixel of thescanned sample, simultaneous Raman imaging of MitoBADY andendogenous cellular biomolecules is readily available from asingle scan. As shown in Figure 4, the subcellular pattern ofMitoBADY, which represents the distribution of mitochondria, isquite distinct from the patterns of endogenous protein and lipid,
as identiﬁed from the amide-I (1686 cm1) and CH2 stretching(2853 cm1) vibrations, respectively. This result demonstrates thatorganelle-speciﬁc Raman probes such as MitoBADY can be used toimage speciﬁc cellular targets simultaneously with intrinsic biomolecules, 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 andapplied it to cellular imaging of mitochondria. The bisphenyl-substituted 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 ofMitoBADY appears in the Raman-silent region of the cell, it is possible

Figure 4. Simultaneous Raman imaging of MitoBADY and endogenous biomolecules 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
conditions: laser wavelength, 532 nm; laser intensity, 2.3 mW/lm2; exposure time,
5 s/line; Scale bar is 10 lm.

H. Yamakoshi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 664–667

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 cellularstructures. 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.
Acknowledgments
The authors thank Dr. Masaya Okada for technical support andhelpful discussions. This work was partly supported by a Grant-inAid for Young Scientist (B) (23710276 to H.Y. and 2410267 to J.A.)
from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Supplementary data
Supplementary data (detailed description of the slit-scanningconfocal Raman microscope, experimental procedures and characterization of all compounds) associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.11.080.
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