A New Infrared Probe Targeting Mitochondria via Regulation of Molecular Hydrophobicity

Article abstract of DOI:10.1021/acs.bioconjchem.8b00845

Herein, we developed a near-infrared (NIR) fluorescent probe for mitochondrial staining based on the NIR fluorochrome, silicon-rhodamine. The hydrophobicity of the fluorescent core was systematically modified by conjugation with 10 different commercial am

Full text of DOI:10.1021/acs.bioconjchem.8b00845

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Article
A new-infrared probe targeting mitochondria
via regulation of molecular hydrophobicity
June Sung, Jun Gi Rho, Gyeong G Jeon, Yeonjeong Chu, Jun
Sik Min, sanghee Lee, Jong Hyun Kim, Wook Kim, and eunha kim
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/
acs.bioconjchem.8b00845 • Publication Date (Web): 18 Dec 2018
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Title: A new-infrared probe targeting mitochondria via regulation of molecular hydrophobicity
Authors: June Sung1,†, Jun Gi Rho1,†, Gyeong G Jeon1, Yeonjeong Chu1,2, Jun Sik Min3, Sanghee Lee2, Jong
Hyun Kim1, Wook Kim1,*, Eunha Kim1,3*
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Affiliations:
1Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea
2Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792,
Korea
3Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon 16499, Korea
*Correspondence to: wookkim21@ajou.ac.kr, ehkim01@ajou.ac.kr
†These authors equally contributed to this work.
Keywords: Silicon rhodamine, Near-IR probe, Fluorescent probe, mitochondria
Abstract:
Herein, we developed a near-infrared (NIR) fluorescent probe for mitochondrial staining based on the NIR
fluorochrome, silicon-rhodamine. The hydrophobicity of the fluorescent core was systematically modified by
conjugation with 10 different commercial amines. The resulting fluorescent compounds exhibited similar
photophysical properties but diverse hydrophobicity. We identified the optimal level of hydrophobicity associated
with high mitochondrial targeting efficiency. In particular, the SiR-Mito 8 probe provided excellent mitochondrial
staining and successfully differentiated the live Hep3B cancer cells from normal L02 cells in vitro.
Keywords
Mitochondria, fluorescent probes, near-IR, silicon rhodamine
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1. Introduction
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Mitochondria provide cellular energy by generating adenosine triphosphate (ATP) via oxidative phosphorylation.
In addition, they play important roles in cell signaling including modulation of ROS production(1), regulation of
apoptotic cell death(2), and maintenance of redox homeostasis(3), innate immunity(4) and autophagy(5).
Consequently, mitochondrial dysfunction results in a number of human conditions ranging from
neurodegenerative diseases(6) to atherosclerosis(7), and diabetes(8).
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Mitochondria are surrounded by a double-membrane, comprising an inner and an outer membrane, and
mitochondrial morphology varies considerably between different cell types. In live cells, mitochondrial
morphology is continuously modified by the combined action of fission and fusion events(9), crucial to maintain
mitochondrial function under cellular stress(10, 11). Therefore, the morphology of mitochondria is dependent on
the cell function and disease states. Recent studies indicated that mitochondrial morphology can be utilized as a
biological marker for the diagnosis of cancer phenotypes and to monitor drug response(12). Consequently, there
is a high demand for fluorescent mitochondrial bioprobes allowing for the investigation of cell signaling and
serving as molecular diagnostic tools.
The large difference in membrane potential (positive in the mitochondrial intermembrane space and negative in
the mitochondrial matrix) across the mitochondrial inner membrane can be efficiently exploited for targeting the
compounds to mitochondria. Therefore, most fluorescent bioprobes for mitochondria are based on compound
conjugation with lipophilic cations (typically triphenyl phosphonium ion)(13), allowing for the efficient transfer
of molecules through lipid bilayers and their accumulation in the mitochondrial matrix.(14) However, systematic
studies addressing the effect of hydrophobicity on the efficiency of mitochondrial targeting by specific
fluorophores are rarely reported. Therefore, developing fluorescent probes to monitor mitochondrial function can
be a difficult task, as it must be preceded by proper optimization. In this context, we reasoned that systematic
modification of fluorophore hydrophobicity could provide an excellent guide for future development of efficient
and versatile mitochondrial fluorescent probes. In addition, considering the growing interest in mitochondria-
specific drug delivery(15-17), this strategy would help developing theragnostic chemical tools, especially for
diseases involving mitochondria.
Herein, we developed a near-infrared (NIR) fluorescent probe for mitochondrial staining. Specifically, the
hydrophobicity of the fluorescent core of a NIR fluorochrome, silicon-rhodamine, was modified by the addition
of 10 different commercial amines. The resulting compounds displayed similar photophysical properties, such as
excitation wavelength (654 ± 4 nm), emission wavelength (667 ± 1 nm), and quantum yield (0.31 ± 0.05), but
diverse cLogP values, ranging from 2.29 to 6.33. By co-staining with a commercial green-fluorescent
mitochondrial probe, we found that probe hydrophobicity had a significant impact on the efficiency of
mitochondria targeting. Among the tested probes, SiR-Mito 8 exhibited excellent mitochondrial staining. In
addition, SiR-Mito 8 successfully distinguished the liver Hep3B cancer cells from normal L02 cells, in vitro.
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2. Results and discussion
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Mitochondria provide cellular energy by utilizing the proton electrochemical gradient potential generated by the
respiratory electron transport chain (ETC) across the mitochondrial membrane. Electron transport, occurring at
the inner mitochondrial membrane, provides the energy required by pumps to translocate proton ions from the
mitochondrial matrix to the intermembrane space (complexes I, III, and IV). Therefore, the most straightforward
approach for targeting mitochondria is the generation of lipophilic cations(14) allowing for probe accumulation
inside the mitochondrial matrix by the electrochemical gradient. To develop a new near-IR mitochondria targeting
bioprobe, we selected SiR-Me(18-20) as the lipophilic cationic fluorochrome. Since, silicon substitution of the
oxygen atom in the rhodamine fluorochrome shifts the emission wavelength of SiR to the NIR range (>680 nm)
preserving the small size of the molecule (MW ≈ 470), we reasoned that SiR-Me would be a suitable fluorochrome
for a systematic evaluation of the effect of probe hydrophobicity on the efficiency of mitochondria targeting.
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Starting with 3-bromoaniline, compound 2 was synthesized by a facile one-pot synthesis process, including
lithiation, silylanization and oxidation of compound 1 (Figure 1)(18-21). The reaction between compound 2 and
lithiated compound 3, followed by acidic deprotection, resulted in the desired SiR-Me compound (compound 4).
Afterward, we conjugated SiR-Me with 10 different commercial amines to generate a library of SiR-Me analogs
(SiR-Mito) with various cLogP values, reflecting different hydrophobicity (Table 1). The resulting library
exhibited a molecular weight range from 470.3 to 554.4 dalton and a cLogP value range from 2.29 to 6.55. We
confirmed that conjugation of the different amines by an amide linkage resulted in probes with different
hydrophobicity but similar photophysical properties such as excitation (654 ± 4 nm) wavelength, emission (667
± 1 nm) wavelength and quantum yield (0.31 ± 0.05).
Figure 1. Synthetic scheme for near-infrared (NIR) fluorescent mitochondria probes based on silicon
rhodamine core. a) Formaldehyde, AcOH, 60 ºC 1.5 h, b) sec-BuLi, SiCl2Me2, THF, -78 ºC, then KMnO4,
acetone, 0 ºC, c) 3, tert-BuLi, THF, -78 ºC, then 6N HCl, 40 ºC d) Di-tert-butyl dicarbonate, DMAP, THF, reflux,
e) PyBOP, DIPEA, amine analogs, DMF, room temperature
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Table 1. Photophysical properties, cLogP value, and correlation coefficient for the colocalization of SiR-Mito
probes with MitoTracker green.
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Mitochondria
cpd.
R
M.W.
Ex/Em (nm)
Φ
clogP
Targetinga
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SiR-Mito 1 2-Hydroxyl ethyl
SiR-Mito 2 Ethyl
486.3
470.3
498.3
532.3
546.3
661/668
662/668
652/665
651/666
651/669
651/668
650/667
651/668
652/666
654/667
0.32
0.40
0.29
0.30
0.29
0.30
0.29
0.33
0.26
0.27
2.29
3.38
4.44
4.82
4.95
5.50
5.94
6.00
6.03
6.33
N.D.
*
SiR-Mito 3 n-Butyl
-
SiR-Mito 4 Benzyl
*
SiR-Mito 5 Phenylethyl
*
SiR-Mito 6 Cyclohexylmethyl 538.3
***
***
***
***
***
SiR-Mito 7 tert-Octyl
554.4
552.3
552.3
554.4
SiR-Mito 8 Cyclooctyl
SiR-Mito 9 Cyclohexylethyl
SiR-Mito 10 2-Methylheptyl
a N.D. : not determined. */**/*** indicate the Pearson coefficient values for colocalization test between the probes
and MitoTracker green. * 0.7 ≤ R < 0.8; ** 0.8 ≤ R < 0.9; *** 0.9 ≤ R
To determine the effect of probe hydrophobicity on the efficiency of targeting to mitochondria, the Pearson
coefficient for colocalization of SiR-Mito probes and MitoTracker green, a commercial fluorescent mitochondrial
bioprobe, was measured (Figure 2). For the colocalization test, human cervical cancer HeLa cells were seeded on
96-well plates and incubated for 1 h with 1 µM of each different SiR-Mito probe and 0.5 µM MitoTracker green,
washed briefly with 1× PBS, and imaged with a LEICA DMi8 fluorescent microscope using the 40× objective.
Interestingly, the alteration of SiR-Mito probe hydrophobicity simply based on conjugation to different amines
dramatically affected mitochondria localization. Pearson coefficient values were significantly different from probe
to probe, ranging from 0.01 to 0.94. For example, the incubation of HeLa cells with SiR-Mito 1, exhibiting a
cLogP value below 3, resulted in the absence of fluorescence signal in the SiR channel (Figure 2). We concluded
that fluorescent conjugates containing SiR with cLogP values lower than 3 were too polar to enter the cells.
Notably, SiR-Mito probes with cLogP values between 3.38 and 4.95 (from SiR-Mito 2 to SiR-Mito 5) were
associated with moderate Pearson coefficients (0.31- 0.79), whereas probes with cLogP values comprised between
5.50 and 6.33 (from SiR-Mito 6 to SiR-Mito 10) resulted in higher Pearson coefficient values (>0.9; Table 1).
Therefore, the latter cLogP range appeared to be the most suitable for developing effective mitochondrial probes
based on SiR fluorochromes. Notably, although the probes from SiR-Mito 6 to SiR-Mito 10 displayed similar
Pearson coefficients (from 0.91 to 0.93), they exhibited different mean of intracellular fluorescence (Figure 3).
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Colocal
SiR
Merged
Colocal
R = 0.93
SiR
Merged
R = 0.01
SiR-Mito 1
SiR-Mito 6
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SiR-Mito 7
SiR-Mito 8
SiR-Mito 9
SiR-Mito 10
SiR-Mito 2
SiR-Mito 3
SiR-Mito 4
SiR-Mito 5
R = 0.91
R = 0.94
R = 0.92
R = 0.93
R = 0.77
R = 0.31
R = 0.70
50 μm
R = 0.79
cLogP
Figure 2. Colocalization test in HeLa cells stained with SiR-Mito probes and MitoTracker Red. HeLa cells
were incubated with cell culture media containing 1 μM SiR-Mito probe, and 0.5 μM MitoTracker green. After 1
h, HeLa cells were washed three times with 1× PBS for and observed with a LEICA DMi8 fluorescent microscope.
Each experiment was observed using 512–542 nm emitter for FITC channel (for MitoTracker Green), and 663–
738 nm exciter, 545–625 nm emitter for Cy5 channel (for SiR-Mito probes). Scale bar: 50 µm.
Figure 3. Fluorescent intensity comparison. Fluorescent intensity of HeLa cells incubated with equal
concentrations of individual probes (Left). Normalized fluorescent intensity (Right).
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The fluorescence intensities produced by equal concentrations (10-5 M in PBS) of the probes SiR-Mito 4 to SiR-
Mito 10 were remarkably similar (Figure 3), demonstrating a significant impact of probe hydrophobicity on
mitochondrial accumulation. Among the probes, SiR-Mito 8 exhibited the highest Pearson coefficient as well as
strongest intracellular fluorescence.
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a.
Mitotracker green
SiR-Mito 8
Merge
b.
Mito-YFP
SiR-Mito 8
Merge
Figure 4. High resolution fluorescence imaging of mitochondria SiR-Mito 8. a) HeLa and Hep3B cells were
incubated with MitoTracker Green (0.5 μM), and SiR-Mito 8 (1 μM) for 1 h. After washing 3 times with fresh
media, fluorescent live cell images were acquired with LEICA DMi8 microscope. (MitoTracker green: FITC
channel, SiR-Mito 8: Cy 5 channel, Scale bar: 15 µm); b) Chang liver cells expressing mitochondrial YFP were
incubated with SiR-Mito 8 (1 μM) for 1 h. After washing 3 times with fresh media, fluorescent live cell images
were acquired with LEICA DMi8 microscope. (Mitochondrial-YFP: Rhodamine channel, Scale bar: 15 µm)
High resolution imaging of HeLa and Hep3B after live cell labeling with SiR-Mito 8 and MitoTracker green
shows excellent colocalization of the two probes (Figure 4a). In addition, high resolution imaging of Chang liver
cells expressing Mitochondrial YFP (Yellow fluorescent Protein) live cell labeling with SiR-Mito 8 further
confirmed high mitochondrial targeting efficiency of SiR-Mito 8 (Figure 4b). To verify the mitochondrial
specificity of SiR-Mito 8 labeling, Hep3B human cancer cells were co-stained with a nuclear and a lysosomal
tracer, in addition to SiR-Mito 8. Unlike other hydrophobic compounds, which are known to accumulate in the
lysosomes of tumor cells(22), live cell imaging with Lysotracker Red demonstrated the high mitochondrial
specificity of SiR-Mito 8 (Figure 5). Therefore, we focused on SiR-Mito 8 for further testing.
It is well known that the membrane potential of mitochondria is significantly higher in cancer cells than in normal
cells.(23, 24) Therefore, we envisioned that SiR-Mito 8 could differentiate cancer cells from normal cells without
involving ligand conjugation.
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Hoechst
Lysotracker
R = 0.50
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SiR-Mito 8
Merged
50 μm
Lysotracker
Figure 5. Co-staining with SiR-Mito 8 and Lysotracker. Liver cancer Hep3B cells were incubated with Hoechst
(0.01 μg/ml), Lysotracker red (1 μM), and SiR-Mito 8 (1 μM) for 1 h. After washing 3 times with fresh media,
fluorescence live cell images were acquired with a LEICA DMi8 microscope (Left). The Pearson coefficient
indicates that SiR-Mito 8 did not localize to other organelle such as lysosome (Right). Scale bar: 50 µm
Figure 6. Cytotoxicity test. HeLa, Hep3B, HepG2 and Raw 264.7 cells were incubated with SiR-Mito 8,
concentration range from 0.25 μM to 10 μM, for 1, 4, 12, and 24 h. Viability of the cells was normalized by
DMSO control.
Before biological investigation of SiR-Mito 8 as a potential NIR fluorescent tracer for mitochondrial staining,
cytotoxicity of the probes was evaluated. HeLa, Hep3B, HepG2 and Raw 264.7 cells were incubated with SiR-
Mito 8 at various concentrations (from 0.25 to 10.0 μM) and cytotoxicity was measured by a mitochondrial
respiration viability assay, confirming the lack of acute cytotoxicity by SiR-Mito 8 upto 24 h time point with
moderate dose (less than 1 μM) (Figure 6). To demonstrate fluorescent tumor imaging, human liver cancer Hep3B
cells and the human hepatocyte line, L02, were incubated with 1.0 μM SiR-Mito 8 for 1 h, washed three times
with cell growth media, and imaged by a LEICA DMi8 fluorescence microscope. We found that the fluorescence
intensity of the cancer cells was significantly higher (5.9 fold) than that of normal cells (Figure 7).
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a.
b.
5
hoechst
SiR-Mito 8
Merged
***
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0
2
g
B
0
n
3
L
a
p
h
e
C
H
50 μm
Cell line
Figure 7. Cellular fluorescence images of Hep3B (liver cancer cells), L02 or Chang Liver cells (normal liver).
a) Cells were incubated with full growth media containing SiR-Mito 8 (1 μM) and Hoechst dye (0.01 μg/ml) for
1 h. The cells were washed 3 times with growth media and observed with a LEICA DMi8 fluorescent microscope.
Cancer cells exhibit higher fluorescence intensity than normal cells. b) Quantification of the fluorescence intensity
of SiR-Mito 8 in L02, Chang liver and in Hep3B cells. Scale bar: 50 µm.
3. Conclusions
In conclusion, we have developed NIR bioprobes selectively staining mitochondria by systematic perturbation
of hydrophobicity of the silicon rhodamine fluorophore. Ten different fluorescent SiR fluorochrome-based
conjugates were synthesized and showed that probe hydrophobicity had a significant effect on the efficiency of
mitochondria-specific staining. The optimal range of cLogP values for mitochondrial targeting by SiR
fluorochromes was from 5.50 to 6.33. Among the 10 probes, SiR-Mito 8 exhibited the highest intracellular
fluorescence intensity, as well as the strongest degree of colocalization with commercial fluorescent
mitochondrial bioprobes in live cells. Moreover, SiR-Mito 8 was successfully applied to in vitro cancer-specific
NIR imaging. We believe that this strategy would be useful in the future development of versatile NIR
mitochondrial probes based on silicon rhodamine fluorophores. In addition, we expect that this study will inspire
the usage of silicon rhodamine fluorochromes as smart drug delivery systems for theragnostics, especially for
cancer.
4. Experimental Procedures
Materials and instruments
All reactions were carried out under an atmosphere of nitrogen or argon in air-dried glassware with magnetic
stirring. Air- and/or moisture-sensitive liquids were transferred with syringe. Organic solutions were
concentrated by rotary evaporation at 25–60 °C at 15–30 torr. All solvents and common materials were
purchased from suppliers and used without further purification. Column chromatography was carried out as
“Flash Chromatography” using Biotage MPLC machine. 1H NMR and 13C NMR data were recorded on an
JEOL ECZ-600R Magnetic Resonance System (600 MHz) in Ajou University. Recorded shifts are reported in
parts per million (δ): Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
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br = broad), coupling constant (J, Hz) and integration. Low resolution mass spectrometry (LRMS) was obtained
by LC/MS system, Finnigan MSQplus Surveyer (Thermo Scientific) or 6120 Quadrupole LC/MS (Agilent
Technologies). The progress of reaction was monitored using thin layer chromatography (TLC) (silica gel 60,
F254 0.25 mm), and components were visualized by observation under UV light (254 and 365 nm) or by
treating the TLC plates with Phosphonolybdic acid (PMA), KMnO4, or ninhydrin followed by heating. Cell
culture reagents including fatal bovine serum, culture media, and antibiotic-antimycotic solution were purchased
from GIBCO. MitoTracker Green, and Hoechst were purchased from Invitrogen. The culture dish and glass-
bottom dish were purchased from CORNING, and SPL. All spectra experiments were performed in a 1×1 cm
quartz cuvette. Fluorescence emission spectra were recorded on JASCO FP-8200 spectrofluorometer, and UV
absorption spectra were recorded on JASCO V-670 spectrophotometer. Absolute quantum yield was measured
by QE-2000 (Otsuka Electronics)
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Chemical synthesis of SiR-Mito probes.
Synthesis of Silicon rhodamine core (compound 4) was carried out as described in previous study(21).
General synthetic procedure.
A solution of compound 4 (1.0 eq), PyBOP (1.3 eq), and DIPEA (3.0 eq) in DMF was stirred for 10 min at room
temperature under argon atmosphere. After 10 min, amine analog (2.0 eq) was added to reaction mixture and
stirred at room temperature for overnight. Reaction mixture was directly purified with 18C reverse column
chromatography with Biotage MPLC.
N-(7-(dimethylamino)-10-(5-((2-hydroxyethyl)carbamoyl)-2-methylphenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-
ylidene)-N-methylmethanaminium (SiR-Mito 1)
1H NMR (600 MHz, Methanol-d4) δ 7.970 (dd, J =7.2Hz, 8.4Hz , 1H), 7.64 (d, J = 3.0 Hz, 1H), 7.54 (d, J = 7.8
Hz, 1H), 7.38 (d, J = 3.0 Hz, 2H), 7.01 (d, J = 9.6 Hz, 2H), 6.79 (dd, J = 9.0 Hz, 2H), 3.69 (t, J = 5.4 Hz, 2H),
3.50 (t, J = 5.4 Hz, 2H), 3.35 (s, 12H), 2.10 (s, 3H), 0.62, 0.61 (s, 6H); 13C NMR (150 MHz, Methanol-d4) δ
155.8, 149.5, 142.1, 141.1, 140.4, 133.2, 131.6, 129.0, 128.8, 128.4, 122.3, 115.4, 61.5, 43.6, 40.9, 19.4, -1.2, -
1.3.
N-(7-(dimethylamino)-10-(5-(ethylcarbamoyl)-2-methylphenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-
N-methylmethanaminium (SiR-Mito 2)
1H NMR (600 MHz, Methanol-d4) δ 7.95 (dd, J = 7.8 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H),
7.38 ( d, J = 3Hz, 2H), 7.06 ( d, J = 9.6 Hz, 2H), 6.79 (dd, J = 9.6Hz, 2H), 3.41 (t, J = 7.2 Hz, 2H), 3.35 (s, 12H),
2.10 (s, 3H), 1.21 (t, J = 6.6Hz, 3H); 13C NMR (150 MHz, Methanol-d4) δ 169.3, 169.0, 155.8, 149.5, 142.1,
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141.0, 140.4, 133.3, 131.6, 129.0, 128.7, 128.4, 122.3, 115.4, 40.9, 35.9, 19.4, 14.9, -1.2, -1.3.
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N-(10-(5-(butylcarbamoyl)-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-
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1H NMR (600 MHz, Methanol-d4) δ 7.95 (dd, J = 7.2Hz, 1H), 7.61 (d, J = 2.4Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H),
7.39 (d, J = 2.4Hz, 2H), 7.06 (d, J = 9.6Hz, 2H), 6.79 (dd, J = 9.6Hz, 2H), 3.37 (t, J = 7.2 Hz, 2H), 3.35 (s, 12H),
2.10 (s, 3H), 1.59 (t, J = 7.2Hz, 2H), 1.40 (q, J = 7.8Hz, 2H), 0.96 (t, J = 7.8Hz, 3H), 0.62 (s, 6H); 13C NMR (150
MHz, Methanol-d4) δ 167.9, 167.6, 154.5, 148.2, 140.8, 139.6, 139.1, 132.0, 130.3, 127.6, 127.4, 127.0, 121.0,
114.0, 39.6, 39.5, 31.3, 19.9, 18.1, 12.8, -2.5, -2.6.
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N-(10-(5-(benzylcarbamoyl)-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-
ylidene)-N-methylmethanaminium (SiR-Mito 4)
1H NMR (600 MHz, Methanol-d4) δ 7.96 (dd, J = 8.4Hz, 1H), 7.62 (d, J = 2.4Hz, 1H), 7.51 (d, J = 8.4Hz, 1H),
7.35 (d, J = 3.0Hz, 2H), 7.29 (m, 5H), 7.03 (d, J = 3.6Hz, 2H), 6.76 (dd, J = 9.6Hz, 2H), 4.53 (s, 2H), 3.32 (s,
12H), 2.07 (s, 3H), 0.58 (s, 6H); 13C NMR (150 MHz, Methanol-d4) δ 169.2, 168.9, 155.8, 149.5, 142.1, 141.2,
140.5, 140.1, 133.1 ,131.8, 129.5, 129.1, 128.8, 128.6, 128.4, 128.2, 122.3, 115.4, 44.6, 40.9, 19.5, -1.2.
N-(10-(5-((cyclohexylmethyl)carbamoyl)-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
3(5H)-ylidene)-N-methylmethanaminium (SiR-Mito 5)
1H NMR (600 MHz, Methanol-d4) δ 7.95 (dd, J = 7.8 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H),
7.38 (d, J = 2.4Hz, 2H), 7.06 (d, J = 10.2 Hz, 2H), 6.79 (dd, J = 9.6 Hz, 2H), 3.34 (s, 12H), 3.21 (d, J = 6.6 Hz,
2H), 2.10 (s, 3H), 1.76 (m, 6H), 1.28 (m, 3H), 0.99 (m, 2H), 0.62 (s, 6H); 13C NMR (150 MHz, Methanol-d4) δ
169.3, 169.1, 155.8, 149.5, 142.1, 141.0, 140.4, 133.4, 131.6, 129.0, 128.7, 128.4, 122.3, 115.4, 47.4, 40.9, 39.3,
32.1, 27.6, 27.0, 19.5, -1.2.
N-(7-(dimethylamino)-5,5-dimethyl-10-(2-methyl-5-(phenethylcarbamoyl)phenyl)dibenzo[b,e]silin-3(5H)-
ylidene)-N-methylmethanaminium (SiR-Mito 6)
1H NMR (600 MHz, Methanol-d4) δ 7.90 (dd, J = 10.2 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.52 (d, J = 1.8 Hz,
1H), 7.38 (d, J = 2.4 Hz, 2H), 7.24 (m, 5H), 7.04 (d, J = 9.6 Hz, 2H), 6.79 (dd, J = 9.6 Hz, 2H), 3.58 (t, J = 7.8
Hz, 2H), 3.35 (s, 12H), 2.90 (t, J = 7.8 Hz, 2H), 2.09 (s, 3H), 0.62 (s, 6H); 13C NMR (150 MHz, Methanol-d4) δ
167.9, 167.7, 154.5, 148.2, 140.8, 139.7, 139.2, 139.0, 132.0, 130.3, 128.5, 128.1, 127.7, 127.3, 127.0, 126.0,
121.0, 114.0, 41.3, 39.6, 35.2, 18.1, -2.5, -2.6.
N-(7-(dimethylamino)-5,5-dimethyl-10-(2-methyl-5-((2,4,4-trimethylpentan-2-
yl)carbamoyl)phenyl)dibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium (SiR-Mito 7)
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1H NMR (600 MHz, Methanol-d4) δ 7.86 (dd, J = 8.4 or 7.2 Hz, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.50 (s, 1H), 7.38
(d, J = 2.4 Hz, 2H), 7.05 (d, J = 10.2 Hz, 2H), 6.78 (dd, J = 10.2 Hz, 2H), 3.35 (s, 12H), 3.16 (m, 6H), 2.91 (s,
1H), 2.08 (s, 3H), 1.94 (s, 2H), 1.85 (m, 6H), 1.48 (s, 6H), 1.01 (s, 9H), 0.61, 0.60 (6H); 13C NMR (150 MHz,
Methanol-d4) δ 169.5, 168.9, 155.8, 149.6, 142.2, 140.5, 140.2, 134.9, 131.4, 129.0, 128.7, 128.4, 122.3, 115.3,
56.8, 51.2, 47.4, 47.4, 43.8, 40.9, 32.6, 31.9, 30.0, 27.4, 27.3, 19.4, -1.2.
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N-(10-(5-((cyclooctylmethyl)carbamoyl)-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
3(5H)-ylidene)-N-methylmethanaminium (SiR-Mito 8)
1H NMR (600 MHz, Methanol-d4) δ 7.95 (dd, J = 8.4 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 3.0 Hz, 1H),
7.38 (d, J = 3.0 Hz, 2H), 7.06 (d, J = 3.0 Hz, 2H), 6.79 (dd, J = 9.6 Hz, 2H), 4.15 (m, 1H), 3.34 (s, 12H), 2.09 (s,
3H), 1.65 (m, 13H), 0.62, 0.61 (6H); 13C NMR (150 MHz, Methanol-d4) δ 169.4, 167.8, 155.8, 149.3, 142.2,
140.8, 140.3, 133.60, 131.5, 129.0, 128.9, 128.4, 122.3, 115.4, 51.6, 40.9, 33.4, 28.1, 26.8, 25.2, 19.4, -1.2.
N-(10-(5-((2-cyclohexylethyl)carbamoyl)-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
3(5H)-ylidene)-N-methylmethanaminium (SiR-Mito 9)
1H NMR (600 MHz, Methanol-d4) δ 7.94 (dd, J = 7.2Hz, 1H), 7.60 (d, J = 1.8Hz, 1H), 7.53 (d, J = 7.2Hz, 1H),
7.38 (d, 3.0Hz, 2H), 7.06 (d, J = 9.6Hz, 2H), 6.79 (dd, J = 9.6Hz, 2H), 3.40 (t, J = 6.6 or 7.8Hz, 2H), 3.34 (s,
12H), 2.10 (s, 3H), 1.70 (m, 7H), 1.50 (d, J = 8.4Hz, 2H), 1.29 (m, 3H), 0.97 (m, 2H), 0.62,0.59 (6H); 13C NMR
(150 MHz, Methanol-d4) δ 168.9, 168.5, 155.4, 149.1, 141.8, 140.6, 140.0, 133.0, 131.2, 128.6, 128.3, 128.0,
121.9, 115.0, 40.5, 38.5, 37.6, 36.4, 34.0, 27.3, 27.0, 19.1, -1.6.
N-(7-(dimethylamino)-5,5-dimethyl-10-(2-methyl-5-(octan-2-ylcarbamoyl)phenyl)dibenzo[b,e]silin-3(5H)-
ylidene)-N-methylmethanaminium (SiR-Mito 10)
1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.4 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H),
7.53 (d, J = 2.4 Hz, 1H), 7.39 (d, J = 2.4 Hz, 2H), 7.07 (d, 9.6 Hz, 2H), 6.80 (dd, J = 9.6 Hz, 2H), 4.15 (m, 1H),
3.34 (s, 12H), 2.10 (s, 3H), 1.30 (m, 10H), 1,21 (d, J = 6.0 Hz, 3H), 0.87 (m, 3H), 0.62, 0.61 (6H); 13C NMR (150
MHz, Methanol-d4) δ 169.3, 168.5, 155.9, 149.6, 142.2, 142.2, 140.9, 140.3, 133.6, 131.6, 129.0, 128.8, 128.4,
122.3, 155.4, 47.3, 40.9, 17.5, 32.9, 30.2, 27.5, 23.6, 21.0, 19.4, 14.4, -1.2, -1.3.
Cell culture and Fluorescent live cell imaging method
HeLa human cervical cancer cells were cultured in Dulbecco’s modified eagle media (DMEM, GIBCO)
containing 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin (GIBCO) at 37 °C in a humidified incubator
with 5% CO2. For the fluorescence microscope imaging, cells were harvested using TrypLETM Express (GIBCO)
and resuspended in fresh culture medium. Harvested cells with a density of 4.0 × 103 cells/well were seeded on a
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96 black well plate (CORNING) and incubated for 24 h. Cells were incubated with SiR-Mito probes (1 μM) and
MitoTracker Green (0.5 μM) for 60 min. After the incubation, cells were washed with 1X PBS for three times and
observed with inverted fluorescence microscope (DMi8, LEICA). Each experiment was observed using a 325–
375 nm exciter, 435–485 nm emitter for DAPI channel, 460–500 nm exciter, 512–542 nm emitter for FITC
channel, 541-551 nm exciter, 565-605 nm emitter for Rhodamine channel, and 663–738 nm exciter, 545–625 nm
emitter for Cy5 channel.
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Lysotracker costaining test
6.0 x 104 human HCC cell line Hep3B cells were seeded on confocal dish and incubated for 2 days. After 2 days,
Hep3B cells were incubated with Hoechst, 1 μM Lysotracker Red, and 1 µM SiR-Mito 8 contained media for 1
h. After the incubation, Hep3B cells were washed with cell culture media for three times and observed with
inverted fluorescence microscope (DMi8, LEICA).
Fluorescent intensity comparison test between normal and cancer cell.
Hep3B, and HepG2 human liver cancer cells were cultured in Dulbecco’s modified eagle media (DMEM, GIBCO)
containing 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin (GIBCO), and L02 human liver normal cells
were cultured in Roswell Park Memorial Institute (RPMI, CAPRICORN) 1640 with L-Glutamine containing 10 %
FBS (GIBCO) and 1% Penicillin (HyClone) at 37 °C in a humidified incubator with 5% CO2. For the fluorescence
microscope imaging, cells were harvested using TrypLETM Express (GIBCO) and resuspended in fresh culture
medium. Harvested cells were seeded on a 96 black well plate (CORNING) with a density of 3.0 × 104 cells/well
for cancer cells, and with a density of 4.0 x 104 cells/well for normal cells. After 24 h incubation. for staining of
mitochondria, cells were incubated with SiR-Mito 8 (1 μM) for 60 min. Cells were washed with cell culture media
for three times and observed with inverted fluorescence microscope (DMi8, LEICA).
Cytotoxicity Test
HeLa human cervical cancer cells, Hep3B and HepG2 human hepatocellular carcinoma cells, and Raw264.7
mouse macrophage cells were seeded into 96-well cell culture plates per well and allowed to adhere to the plates
overnight. The cells were incubated with cell culture media containing each concentration of SiR-Mito 8 (0.25
μM to 10 μM) for 1, 4, 12 or 24h. after incubation, CellTiter 96® Aqueous One Solution Cell Proliferation Assay
(Promega) was added into each well. After 2h, 490 nm absorption was measured with Cytation 3 microplate reader
(BioTek). Cytotoxicity of SiR-Mito 8 was tested in triplicate in a single experiment, with each experiment being
repeated at least three times.
Fluorescent high-resolution imaging in live cell
HeLa, and Hep3B cells were cultured in Dulbecco’s modified eagle media (DMEM, GIBCO) containing 10%
fetal bovine serum (FBS, GIBCO) and 1% penicillin (GIBCO) at 37 °C in a humidified incubator with 5% CO2.
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For the fluorescence microscope imaging, cells were harvested using TrypLETM Express (GIBCO) and
resuspended in fresh culture medium. Harvested cells were seeded on a confocal dish with a density of 4.0 × 104
cells/well. After 24 h incubation, cells were incubated with SiR-Mito 8 (1 μM) and Mitotracker green (0.5 uM)
for 60 min. Cells were washed three times with 1X PBS and observed with inverted fluorescence microscope
(DMi8, LEICA).
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Plasmid transfection.
Overexpression plasmid for Mitochondria-YFP was obtained from Catholic University (Seoul, Korea) and
transfected into Chang liver cells. Stable clones expressing Mitochondria-YFP was selected using 0.25mg/ml
G418.
Costaining test of SiR-Mito 8 with Mitochondria-YFP
Mitochondria-YFP infected Chang liver cells were cultured in Dulbecco’s modified eagle media (DMEM,
GIBCO) containing 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin (GIBCO) at 37 °C in a humidified
incubator with 5% CO2. For the fluorescence microscope imaging, cells were harvested using TrypLETM Express
(GIBCO) and resuspended in fresh culture medium. Harvested cells were seeded on a confocal dish with a density
of 4.0 × 104 cells/well. After 24 h incubation, cells were incubated with SiR-Mito 8 (1 μM) for 60 min. Cells were
washed three times with 1X PBS and observed with inverted fluorescence microscope (DMi8, LEICA).
Notes
The authors declare no competing financial and non-financial interests.
Supporting Information
1H NMR spectrums, 13C NMR spectrums, abs spectrums and emission spectrums of SiR-Mito probes.
Acknowledgments
This work was supported by Basic Research Program (2009-0093826, 2016R1E1A1A01941213 and
2016R1C1B2014699) through the National Research Foundation of Korea (NRF) funded by the Korean
Government (Ministry of Science and ICT) and by the Ajou University research fund.
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