Targetable and fixable rotor for quantifying mitochondrial viscosity of living cells by fluorescence lifetime imaging

Article abstract of DOI:10.1039/c6tb02524b

It is meaningful to accurately quantify the changes in local viscosity within the mitochondria of living cells, because viscosity influences mitochondrial network organization and metabolite diffusion. Although many molecular probes targeting mitochondria have been reported, almost all of them are not fixed to the mitochondria. Thus, they may not be suitable for sensing in abnormal mitochondria with lowered potential. In order to monitor viscosity in all mitochondria, no matter their working or health status, we develop the first fixable BODIPY (boron-dipyrromethene) rotor, named Vis-A. Vis-A contains an aldehyde group as an anchor to react with amino groups of proteins, which make it immobilizable in mitochondria. Vis-B, the reference compound without such anchor unit, is also synthesized. Both Vis-A and Vis-Bshow excellent mitochondrial targetability, as good as the commercially available mitochondrial dye (Mito Tracker Deep Red). However, when cells are chemically treated to decrease the mitochondrial potential, only Vis-A continues emitting strong fluorescence in mitochondria, but the signals of Vis-B and Mito Tracker Deep Red completely disappear. This comparison confirms that Vis-A not only specifically localizes in mitochondria, but also can be stably retained there. Then, Vis-A is applied to detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A, the changes in viscosity under typical pathological conditions (i.e., treatment with rotenone and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) for mitochondria are monitored by FLIM.

Full text of DOI:10.1039/c6tb02524b

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Targetable and Fixable Rotor for Quantifying Mitochondrial
Viscosity of Living Cells by Fluorescence Lifetime Imaging
Xinbo Song,a Ning Li,a Chao Wang,a and Yi Xiaoa*
Received 00th January 20xx,
Accepted 00th January 20xx
It is meaninful to accurately quantify the changes of local viscosity within mitochonria of living cells, because viscosity
influeneces the mitochondrial network organization and the metabolite diffusion. Although many molecular probes
targeting mitochondria have been reported, almost all of them are not fixed to the mitochondria. Thus, they may not be
suitable for sensing in abnormal mitochondrial with lowered potential. In order to monitor viscosity in all mitochondria, no
matter which are in their working or health status, we develop the first fixable BODIPY (boron-dipyrromethene) rotor,
named Vis-A. Vis-A contains an aldehyde as anchor to react with aminos of proteins, which make it immobilizable in
mitochondria. Vis-B, the reference compound without such anchor unit, is also synthesized. Both Vis-A and Vis-B, show
excellent mitochondial targetability, as good as the commercially available mitochondiral dye (Mito Traker Deep Red).
However, when cells are chemically treated to decrease the mitochondrial potential, only Vis-A continues emitting strong
fluorescence in mitochondria, but the singals of Vis-B and Mito Traker Deep Red completely disappear. This comparison
confirms that Vis-A not only specifically localize in mitochondria, but also can stably retend there. Then, Vis-A is applied to
detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A,
the changes of viscosity under typical pathological conditions ( i.e, treatment with rotenone and carbonylcyanide-m-
chlorophenylhydrazone, (CCCP) for mintochodrial are monitored by FLIM.
DOI: 10.1039/x0xx00000x
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diffuse away from mitochondria for the lowering of the
attraction. Thus, almost all those mitochondria targeted probes
might be unsuitable for the studies on abnormal mitochondria,
Introduction
Mitochondria, known as power houses, play critical roles
in cellular viability and the overall health. They are in charge of
energy generation through the degrade of nutrients, which is the
prerequisite for cells to maintain homeostasis and function
properly.1 Additionally, if mitochondrial microenvironment
factors deviate from normal levels, mitochondria may go wrong
to induce cells’ malfunctions or even death.2ꢀ6 Therefore, it is
important to detect microenvironment changes in mitochondria.
After years of efforts from many research teams, quite a
few chemical probes have been developed to sense and image
environmental factors in the mitochondria, such as metal ion,7ꢀ
14, redox environment,15ꢀ29 polarity,30ꢀ33 viscosity,34ꢀ37 pH,38ꢀ42
and temperature.43 Almost all these probes contain positively
charged unit, which enables them to be accumulated in
mitochondria, due to the attraction by the negative
mitochondrial potential.44ꢀ48 However, there still exists one big
challenge. It is well known, under some pathological
conditions, mitochondrial potential will be considerably
decreased. Under these circumstances, probe molecules will
since they cannot stably retain there. In principle, this critical
problem can be solved by fixation of probes in mitochondria by
the formation of chemical bonds. But, to the best of our
knowledge, only two cases on this topic have been reported
recently. In 2014, Lee et al developed a fixable mitochondrial
pH probe.38 In 2015, our group reported a fixable polarity
probe, which was used for quantitative detection of
mitochondrial membrane potential in pathological conditions.30
Scheme 1. Working Principle of Mitochondrial Viscosity Probes.
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Scheme 2. Synthetic routes for Vis-A and Vis-B. (I) 4-Hydroxybenzaldehde/pyrrole/TFA, rt, 12 h, 94%; (II) DDQ, DCM, rt, 1 h, 90%; (III) BF3-Et2O/DIEA, PhMe, 70 oC, 0.5
h , 75%; (IV) 1,4-bis(chloromethyl)benzene/K2CO3, MeCN, reflux, 15 min, 90%; (V) 4-Bromobenzaldehyde/NaI/Pd/C, DMF, 160oC, 8 h, 35%; (VI) e or f/NaI, MeCN,
reflux, 6 h, 95% or 45%.
Figure 1. A: The absorption of Vis-A in methanol; B: Fluorescence spectra of Vis-A in methanol/glycerol mixtures; C: The relationship between fluorescence intensity
and the viscosity; D: The absorption of Vis-B in methanol; E: Fluorescence spectra of Vis-B in methanol/glycerol mixtures; F: The relationship between fluorescence
intensity and the viscosity.
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Figure 2. A: The fluorescence lifetime spectra at 520 nm for 3
relationship of Vis-A between the fluorescence intensity and the viscosity; C: The fluorescence lifetime of Vis-A responses to different polar solution. D: The
fluorescence lifetime spectra at 520 nm for 3 Vis-B in methanol/glycerol mixtures with varying proportions to adjust the viscosity; E: The relationship of Vis-B
ꢁM Vis-A in methanol/glycerol mixtures with varying proportions to adjust the viscosity; B: The
ꢁ
M
between the fluorescence intensity and the viscosity; F: The fluorescence lifetime of Vis-B responses to different polar solution.
In this work, we focus on probes for mitochondrial with media viscosity, which provides the basis for monitoring
viscosity that is known to influence the signalling interaction of mitochondrial viscosity via fluorescence lifetime imaging
the biomolecules and chemicals. Abnormal viscosity variation (FLIM). Last but not the least, probe Vis-A contains aldehyde,
in the mitochondrial matrix may induce changes in which can react with an amino of the proteins and be
mitochondrial network organization, and further influence immobilized permanently on the proteins
(Scheme 1).
metabolite diffusion, which, consequently, are related to According to one of our recent studies, the first case of reactive
cellular aspects of many diseases and malfunctions, such as cell aldehyde conjugated to a triphenylphosphonium moiety proved
malignancy, atherosclerosis, diabetes, and Alzheimer’s successful in targeting and fixing
disease.49ꢀ51 So far, there is not any report on a viscosity probe mitochondrial proteins.30
a probe onto the
immobilized the mitochondria. Therefore, our aim is to design a
practical probe for quantifying viscosity in all kinds of efficiently. The synthesis procedures are illustrated in Scheme
mitochondria, no matter their working or health status. . The reaction conditions and structural characterization are
The probes, Vis-A and Vis-B, are synthesized very
2
Based on the above consideration, we develop the first described in the Experimental section.
fixable probe for mitochondrial viscosity. As shown in Scheme
Photophysical properties of Vis-A and Vis-B
1, the key innovation of this viscosity probe, Vis-A, compared
with several previously reported ones is its possession of a
To characterize the spectral properties of the sensors, we
reactive aldehyde that can form stable covalent bond with first study the absorption and the emission of the probes in
aminos in proteins. In this paper, we confirm the applicability methanol. Their maximum absorption of the wavelength is at
of Vis-A
,
in terms of its viscosity responsiveness, 501 nm and their strongest fluorescence wavelength is at 517
mitochondriaꢀtargeting specificity, and its stable retention in nm in methanol. There is a very large change of the fluorescent
abnormal mitochondria. And we also apply Vis-A to monitor intensity of the probes at various viscosities of the solvents
the viscosity changes under typical pathological conditions.
(Figure 1). The probes show very weak fluorescent intensity in
lowꢀviscosity media and they show highly fluorescent intensity
in viscous glycerol. These results are attributable to viscosityꢀ
dependent rotation of the C−C bond between the BODIPY and
the phenyl unit, where the rotation through the C−C bond is
restricted in the presence of a highly viscous solvent, such as
glycerol, and the excited energy is reserved for emission
without nonradioactive energy dissipation throughout the
rotation. And the relationship between the fluorescence
intensity and viscosity is showed in figure 1C and 1F. The
fluorescence intensity is influenced by concentration of the
Results and discussion
Design and synthesis
There are several considerations in the molecular design of
probe Vis-A. The cationic triphenylphosphonium is expected to
facilitate the selective accumulation of the probe in
mitochondria.44 And the rotor unit provides sensitivity toward
the viscosity, due to the free rotation around the single bond
connecting the BODIPY core and the phenyl group.34, 52 The
fluorescence lifetime of the probe shows the linear relationship
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probes and lasers. So we investigate the relationship between solvent components. As shown in figure 2C, in all the waterꢀ
the fluorescence lifetime of the probes and the viscosity. dioxane mixed solvents, fluorescence lifetimes of Vis-A are
The fluorescence lifetimes of both the probes are measured very similar (around 0.68ns). We also measure fluorescence
in a series of viscosity gradient buffers, which are composed of lifetime in lowꢀpolarity solvents i.e. toluene and
methanol and glycerol in various proportions. Interestingly, the dichloromethane, and the similar values are detected. These
fluorescence lifetime of Vis-A at 520 nm increases gradually results indicate that fluorescence lifetime of Vis-A is insensitive
from 0.6ns to 5.9ns as the viscosity increases from 0.6cp to to the variation of solvent polarity. Interestingly, when glycerol
360cp (Figure 2A). And the fluorescence lifetime of Vis-B at is used as solvent, the fluorescence lifetime value becomes very
520nm increases gradually from 0.6ns to 6.3ns as the viscosity large (7.0ns). The longer lifetime in glycerol can be attributed
increases from 0.6cp to 360cp (Figure 2D). In particular, there to the high viscosity of this solvent and is not related to its
are good linear relationships observed between fluorescence polarity. The same conclusion for Vis-B can also be made,
lifetimes and solvent viscosity of the both probes (R2 =0.99) according to figure 2F. The excellent selectivity of Vis-A and
(
Figure 2B, and 2E).
Vis-B for viscosity is inherited to the parent BODIPY rotor.
Selectivity is the most important requirement for all kinds Therefore, our probes have great potential in viscosity detection
of detection methods. For some viscosity probes, their in complex biological environments.
fluorescence properties were found to be also highly sensitive
Localization stabilities of Vis-A and Vis-B in mitochondria
to solvent polarity.53 This will be problematic for the
applications in the complex intracellular environment, because
Colocalization studies of Vis-A and Vis-B with Mito
it will be hardly possible to distinguish the effect of viscosity to Tracker Deep Red (Commercial mitochondrial tracker) have
fluorescence from that of polarity. In order to confirm that the been conducted in 7721 cells, Hela cells, and MCFꢀ7 cells. As
fluorescence lifetimes of our probes Vis-A and Vis-B are demonstrated in figure
3, Vis-A or Vis-B emits green
selectively responsive to viscosity changes but independent on fluorescence signals in channel 1, while the commercial
polarity, we decide to carry out a measurement in several mitochondrial tracker emits red signals in channel 2. The
solvents with different polarity. According to a literature,54 in merged images (Figure 3C and 3G) indicate that the two
the mixtures of water and 1,4ꢀdioxane, a series of polarity channel images overlap very well, confirming that the both
gradient systems can be obtained by tuning the ratio of the two probes can specially localize in the mitochondria of the living
Figure 3. Colocalization imaging studies of Vis-A and Vis-B in 7721 cells. A Vis-A Channel 1: λex=488nm, λem=500-550nm. B Mito Tracker Deep Red Channel 2:
λex=635nm, λem=655-755nm. C overlay of A and B (Pearson coefficient 0.97). E: Vis-B Channel 1: λex=488nm, λem=500-550nm. F Mito Tracker Deep Red Channel 2:
λex=635nm, λem=655-755nm. G overlay of E and F (Pearson coefficient 0.92). D and H: Intensity profile of ROIs across the cells.
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Figure 4. Imaging studies of Vis-A and Vis-B stain in 7721 cells. A, and B: The cells are treated with Vis-A (2μM) and Mito Traker Deep Red (0.5μM) for 90 min, washed
three times by PBS. C and D: The cells are treated with Vis-A (1μM) and Mito Traker Deep Red (0.5μM) for 90 min, washed three times by PBS, cultured with four
percent formaldehyde solution for 1 hours at 4oC, and then washed by the solution (ethanol : PBS = 1 : 5) for three times. A and C Vis-A Channel 1: λex=488nm,
λem=500-550nm. B and D Mito Tracker Deep Red Channel 2: λex=635nm, λem=655-755nm; E and F: The cells are treated with Vis-B (1μM) and Mito Traker Deep Red
(0.5μM) for 90 min, washed three times by PBS. G and H: The cells are treated with Vis-B (1μM) and Mito Traker Deep Red (0.5μM) for 90 min, washed three times by
PBS, cultured with four percent formaldehyde solution for 1 hours at 4oC, and then washed by the solution (ethanol : PBS = 1 : 5) for three times. E and G Vis-B
Channel 1: λex=488nm, λem=500-550nm. F and H Mito Tracker Deep Red Channel 2: λex=635nm, λem=655-755nm.
set of colocalization imaging experiments under extreme
conditions, as demonstrated in figure 4. 7721 cells are treated
and cultured with four percent formaldehyde solution, which
results in cell death and the complete elimination of
mitochondrial potential.
As is expected, before and after the treatment, Vis-A
exhibits strong intracellular fluorescence (Figure 4A and 4C),
which means the decrease of mitochondrial potential will not
drive Vis-A away from mitochondria. In contrast, the initially
strong fluorescence signals of Vis-B and Mito Tracker Deep
Red decrease remarkably (Figure 4G) or even disappear
(
Figure 4H) after the same treatment, indicating that they no
longer stay in the mitochondria of the dead cells. And we also
perform the same imaging experiments on other type of cells,
e.g. MCFꢀ7 and Hela cells (see supporting information) treated
with formaldehyde. Again, the stable retention of Vis-A in
mitochondria is observed. Since the only structural difference
between Vis-A and Vis-B is the presence or absence of one
aldehyde group, it is logical to ascribe the immobilization of
Vis-A in mitochondria to this anchoring group.
In order to further prove the high efficiency of Vis-A to
react with mitochondrial proteins, flow cytometry analysis is
used to quantitatively evaluate fixationꢀinduced changes of
fluorescence intensity. We divide the flow cytometry
experiments into two sets. First one is the control experiment
without formaldehyde fixation, in which MCFꢀ7 cells are
incubated with Vis-A for 90 min and then washed three times
by PBS. In second set, the cells are stained with the Vis-A for
90 min, washed three times by PBS, then treated with four
percent formaldehyde solution for 1 hours at 4oC, and finally
washed by PBS three times. Then flow cytometry analyses are
conducted for both groups of cells. The changes of intracellular
fluorescence intensity are recorded in figure 5. Surprisingly,
the fluorescence intensity of the formaldehydeꢀfixed cells is not
Figure 5. Flow cytometric analysis of MCF-7 cells. a, b, and c: The fluorescence
intensity of the cells which are treated with Vis-A (4μM) for 90 min, washed
three times by PBS. d, e, and f: The fluorescence intensity of the cells which are
treated with Vis-A (4μM) for 90 min, washed three times by PBS, cultured with
four percent formaldehyde solution for 1 hours at 4oC, and then washed by PBS
for three times.
cells. The changes in the intensity profile of linear regions of
interest (ROIs) (Probes and commercial tracker) tend toward
synchronization (Figure 3D and 3H). And the high Pearson’s
coefficients are obtained (Vis-A: Pearson coefficient 0.92 in
7721 cells; Vis-B: Pearson coefficient 0.97 in 7721 cells). The
other images obtained in the colocalization studies are in the
supporting information. Above investigation results confirm
that both Vis-A and Vis-B target mitochondria specifically,
because of the attraction from negative potentials in the
mitochondria of the living cells.
As we have mentioned in the introduction section, an ideal
mitochondrial probe should not only work in the normal
mitochondria with relatively high potentials, but also it should
continue working during the pathological processes in which
the potential might be lowered considerably. To verify that Vis-
A
will permanently retain in mitochondria, we carry out another
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Figure 6. Imaging studies of Vis-A and Vis-B stain in 7721 cells. A: Fluorescence imaging of Vis-A in 7721 cells. B: Fluorescence life time imaging of Vis-A in 7721 cells.
C: Fluorescence lifetime distribution histogram for B. D: The fluorescence decay curve of point D in image B. E: Fluorescence imaging of Vis-B in 7721 cells. F:
Fluorescence lifetime imaging of Vis-B in 7721 cells. G: Fluorescence lifetime distribution histogram for E. H: The fluorescence decay curve of point H in image F. 7721
cells are treated with Vis-A (1μM) for 90 min, washed three times by PBS. 7721 cells are treated with Vis-B (2μM) for 90 min, washed three times by PBS.
Figure 7. 7721 cells are treated with Vis-A (2μM) for 90 min, washed three times by PBS. A, C, E, and G: Fluorescence imaging of Vis-A in 7721 cells. B(A), D(C), F(E),
and H(G): Fluorescence life time imaging of Vis-A in 7721 cells. A and B: The control group. C and D: The cells are stimulated by rotenone (10μM) for 8.5h. E and F: The
cells are stimulated by rotenone (10μM) for 18h. G and H: The cells are stimulated by CCCP (100ꢁM) for 3h.
lower but is even stronger than that of untreated cells. Our
Monitoring normal and abnormal mitochondrial viscosity
In combination FLIM Vis-A and Vis-B are reliable tools for
quantifying the local viscosity within mitochondria. Figure 6
shows FLIM images of 7721 cells, in which fluorescence
lifetime in the mitochondria has been mapped with considerable
explanation is that, in the fixed cells, the local viscosity of
mitochondria is much higher than that of the normal living
cells. The fluorescence enhancement caused by higher viscosity
is more pronounced than the possible fluorescence decrease
resulted from the washedꢀout of the unfixed probes. Although
above flow cytometry analysis fails in determining the probeꢀ
fixation percentage, it at least indirectly proves that the dye
fixation efficiency is high sufficiently for further fluorescence
imaging investigations.
,
spatial resolution. In the FLIM image 6B, the average
fluorescence lifetime of Vis-A is 2.07ns, which is obtained
from the fluorescence lifetime distribution histogram. Thus the
average viscosity around Vis-A in mitochondria is determined
to be about 95cp, according to our calibration graph in figure
2B. And the average fluorescence lifetime of Vis-B is 1.62ns
(
Figure 6F and 6G), indicating the average viscosity around
Vis-B is about 63cp. It is interesting that Vis-A and Vis-B
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possessing the same rotor unit give different values of mitochondria increase greatly. Thus, under the above
mitochondrial viscosity. We explain this difference by the pathological conditions, our probe Vis-A demonstrates the
different location: Vis-A is covalently bound to proteins, while applicability to quantify the changes of mitochondrial viscosity
the unbound Vis-B is located in the lipid bilayer of and monitor mitochondrial morphology simultaneously.
mitochondrial inner membranes. It is possible that
macromolecular structures of proteins contribute to a larger
Conclusion
viscosity reflected by the longer fluorescence lifetime of Vis-A.
Also noticeable is the wider fluorescence lifetime
distribution histogram of Vis-A (Figure 6C) than that of Vis-B
(Figure 6G). This might be due to the existence of many
aminos in different sites of proteins with different structures,
which produces quite different local viscosity around the bound
Vis-A molecules. The above regulations obtained in 7721 cells
are also observed when Vis-A and Vis-B are used to label other
type of cells e.g. MCFꢀ7 cells (supporting information).
In conclusion, we have developed Vis-A, a new probe
that can specifically target mitochondria and quantify local
viscosity by its fluorescence lifetime. Compared with other
viscosity probes, Vis-A contains a formyl group to react with
aminos of proteins. For this reason, Vis-A can be permanently
immobilized into mitochondria via forming covalent bond. This
feature of Vis-A is helpful to overcome the nonꢀfixed probes’
problem of unstable mitochondrial localization. Such advantage
of Vis-A is confirmed in the applications under two typical
pathological conditions that damage mitochondria. Cells are
stimulated with rotenone and CCCP. In both cases, apparent
increases in mitochondrial viscosity are quantified by FLIM
imaging with the aid of Vis-A. And it is believed that Vis-A
provides a strategy in inspecting mitochondrial viscosity and in
diagnosis of mitochondria related diseases.
The FLIM images (Figure 6B and 6F) exhibit high spatial
resolution almost as good as corresponding confocal images
(
Figure 6A and 6E). The fluorescence lifetime distribution is
so clearly mapped that it is feasible to determine the
fluorescence lifetimes/viscosities of single mitochondria. For
example, we randomly choose two regions (
D and H) in cells,
and obtained their fluorescence decay curves (Figure 6D and
6H) that are as precise as those previously detected in solution
(
Figure 2A and 2D).
Experimental section
As we mentioned, we hope to monitor the viscosity
changes in the abnormal mitochondria. We have proved that,
the immobilized Vis-A will permanently retain and continue
emitting in mitochondria. Naturally, our next work is to
confirm applicability of Vis-A in the pathological processes
that mitochondria undergo damages. For this sake, rotenone, a
chemical affecting cell respiration by inhibiting the transfer of
electronic chain in mitochondria, is used to stimulate 7721
cells.55ꢀ59 During a 8.5h period of stimulation using rotenone
(10 ꢁM), according to the fluorescence lifetime distribution
histogram, FLIM images reveal that the fluorescence lifetime in
mitochondria of 7721 cells changes from 2.0ns to 2.45ns
Culture cells
MCFꢀ7 cells, 7721 cells, and HeLa cells are obtained from
Institute of Basic Medical Sciences (IBMS) of Chinese
Academy of Medical Sciences (CAMS). All cell lines are
maintained under standard culture conditions (atmosphere of
5% CO2 and 95% air at 37 °C) in RPMI 1640 medium or
DMEM medium, supplemented with 10% FBS (fetal bovine
serum). The cells is used after the cells in the exponential phase
of growth on 35 mm glass bottom culture dishes (Φ 20 mm) for
1 day.
Synthesis
(
Figure 7B and 7D), which means that the average viscosity of
mitochondria around the probe Vis-A increases considerably.
And after the cells are incubated with rotenone for 18h, the
average fluorescence lifetime further increases to 2.73ns
All chemicals are obtained from commercial suppliers and used
without further purification. The 400 (1H) MHz NMR and 100
(13C) MHz NMR spectra are registered at room temperature on
a 400 MHz spectrometer using perdeuterated solvents as
(
Figure 7F), indicating that further extension of treatment time
will result in a slower increase in mitochondrial viscosity.
And the other stimulating agent is carbonylcyanideꢀmꢀ
chlorophenylhydrazone (CCCP), which is used to fast abolish
the inner mitochondrial membrane potential, as this agent is one
of the representative uncouplers for mitochondriaꢀrelated
pathology and pharmacology.60 After the cells are stained with
CCCP (100ꢁM) for 3h, FLIM images indicate that the
fluorescence lifetime in mitochondria of MCFꢀ7 cells increases
from 2.09ns to 2.22ns, which shows that the average viscosity
internal standard. Images are acquired with
microscopy from OLYMPUS.
General procedure for the synthesis of the probe and the
probe’s derivatives
a confocal
Preparation of a 4ꢀHydroxybenzaldehde (6 g, 49 mmol) is
added to freshly distilled pyrrole under the protection of
argon.61 And then trifluoroacetic (0.2 mml) is dropped in the
solution. The reaction mixture is stirred at room temperature for
12h. Excess of pyrrole is distilled under reduced pressure to
give the black liquid, which is purified by silica gel flash
chromatography using CH2Cl2/CH3OH (200:1 to 50:1) as
eluent to afford compound a (11g, yields 94%). Compound a
obtained as a grey solid is used for the next step without further
purification.
of mitochondria around the probe Vis-A increases
(Figure
7H). Although CCCP induced the fluorescence lifetime of the
probes is not so pronounced as rotenoneꢀinduced, the former is
faster. And it is also observed that CCCP stimulation causes a
remarkable morphological change. As shown in figure 7G and
7H, mitochondria turn from filamentary organizations to
punctual structures, and it seems that the number of
Preparation of b: Compound a (1g, 4.2 mmol) is dissolved in
CH2Cl2 (20 mL) and then DDQ (1.1g 5.0mmol) is added to the
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solution. The mixture is stirred for 1h. Filter out solid and then 7.53 (d,
the solvent is removed under reduced pressure to give the crude = 8.1 Hz, 2H), 7.08 (d,
black product, which is purified by silica gel flash 2H), 6.54 (d, = 2.5 Hz, 2H), 5.09 (s, 2H).
chromatography using CH2Cl2/CH3OH (200:1 to 40:1) as
13C NMR (126 MHz, CDCl3) δ 190.78 (s), 160.95 (s),
eluent to afford compound b (0.9g, yields 90%). Compound b 147.14 (s), 143.58 (s), 140.21 (d, = 2.7 Hz), 136.95 (d, = 4.2
obtained as a grey solid is used for the next step without further Hz), 135.42 (dd, = 10.0, 6.6 Hz), 134.84 (s), 134.63 (d,
purification. 9.9 Hz), 132.45 (s), 132.09 (d, = 5.5 Hz), 131.36 (s), 130.34
Preparation of c: Compound b (4.23 mmol, 1 g) is added to a (d, = 12.7 Hz), 127.81 (d, = 3.3 Hz), 126.81 (s), 124.56 (s),
15 mL toluene. The mixture is heated to 70 Nꢀ 123.89 (s), 118.37 (s), 117.24 (s), 116.56 (s), 114.93 (s), 69.60
Ethyldiisopropylamine (21.6 mmol, 3.7mL) is dropped to the (s), 30.38 (s).
solution. After the solution is stirred for 30min, ethyl ether Preparation of Vis-B: Compound
boron fluoride is dropped to the solution. And the reaction is mmol), NaI (70 mg, 0.54 mmol) and triphenylphosphine (40
stirred at 70 for 2h. Then the solvent is removed under mg, 0.16 mmol) are dissolved in 3mL acetonitrile under
J
= 8.7 Hz, 2H), 7.29 (dd,
J
= 8.2, 2.1 Hz, 2H), 7.25 (d,
J
J = 8.7 Hz, 2H), 6.95 (d, J = 4.0 Hz,
J
J
J
J
J =
J
J
J
℃
.
d
(23 mg, 0.054
℃
reduced pressure to give the crude black product, which is nitrogen protection. The mixture is refluxed for 40min. Then
purified by silica gel flash chromatography using the acetonitrile is removed under reduced pressure. The mixture
CH2Cl2/CH3OH (200:1 to 80:1) as eluent to afford compound c is
(0.9g, yields 75%). (gradient:CH2Cl2/CH3OH 40:1
1HNMR (400 MHz, CDCl3) δ 6.55 (m, 2H), 6.97 (m, 4H), 7.45 80%). m/z (FTMS+p ESI): Calcd [M+] for C41H33BF2N2OP+,
purified
by
column
chromatography
→15:1) to obtain Vis-B (34 mg,
(d, 2H), 7.90 (s, 2H, ).
Preparation of d: Compound c (150 mg, 0.53 mmol) and 1, 4ꢀ
649.2386, found 649.2382.
1H NMR (500 MHz, CDCl3) δ 7.90 (s, 2H), 7.75 (ddd,
J
=
bis(chloromethyl)ꢀbenzene (186mg, 1.06 mmol) are dissolved 19.9, 10.3, 6.9 Hz, 9H), 7.63 (td,
in acetonitrile (5 mL). Potassium carbonate (146 mg, 1mmol) is = 8.7 Hz, 2H), 7.25 (d, = 8.0 Hz, 2H), 7.19 (dd,
added to the solution and the solution is refluxed for 40min. Hz, 2H), 7.06 (d, = 8.7 Hz, 2H), 6.95 (d, = 4.0 Hz, 2H), 6.54
The solvent is removed under reduced pressure to give the (dd, = 4.1, 1.7 Hz, 2H), 5.32 (d, = 14.4 Hz, 2H), 5.10 (d,
crude product, which is purified by silica gel flash 1.5 Hz, 2H).
chromatography using CH2Cl2/hexane (1:1) as eluent to afford
13C NMR (126 MHz, CDCl3) δ 160.95 (s), 147.18 (s),
compound d (202mg, yields 90%). m/z (TOF MS EI+): Calcd 143.48 (s), 136.82 (d, = 4.0 Hz), 135.14 (d, = 2.9 Hz),
= 9.8 Hz), 132.41 (s), 131.89 (d, = 5.5
Hz), 131.37 (s), 130.23 (d, = 12.6 Hz), 127.72 (d, = 3.3 Hz),
J
= 7.9, 3.5 Hz, 6H), 7.51 (d,
J
J
J = 8.2, 2.3
J
J
J
J
J =
J
J
[M+] for C23H18BClF2N2O 422.1169, found 422.1176.
1H NMR (500 MHz, CDCl3) δ 7.92 (s, 2H), 7.54 (d,
134.80 (s), 134.44 (d,
J
J
J
=
J
J
8.7 Hz, 2H), 7.49 – 7.43 (m, 4H), 7.11 (d,
(d, = 4.0 Hz, 2H), 6.54 (d, = 3.0 Hz, 2H), 5.16 (s, 2H), 4.61 (s), 29.67 (s).
(s, 2H).
J = 8.6 Hz, 2H), 6.96 126.69 (s), 118.36 (s), 117.90 (s), 117.22 (s), 114.98 (s), 69.60
J
J
13C NMR (126 MHz, CDCl3) δ 161.13 (s), 147.27 (s), 143.51
(s), 137.61 (s), 136.56 (s), 134.86 (s), 132.43 (s), 131.35 (s),
128.98 (s), 127.80 (s), 126.71 (s), 118.30 (s), 114.91 (s), 69.85
(s), 45.80 (s).
Preparation of f: 4ꢀBromoacetophenone (1 g, 5.4 mmol) ), 5%
(w/w) palladium (287.3mg, 0.27mmol ), triphenylphosphine
(3.537 g, 13.5 mmol ) and NaI (1.62 g, 10.8 mmol ) are added
to anhydrous DMF under nitrogen protection. The mixture is
stirred at 160 °C for 8 h. The reaction mixture is cooled to room
temperature. And the reaction mixture is filtrated to remove
insoluble impurities, then the DMF is removed under reduced
pressure. The residue is purified by silica gel column
chromatography using eluent hexanes/CH2Cl2 (3/1, v/v). A
white solid is obtained (620 mg, 39.6 %).
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (nos. 21174022, 21376038, 21421005,
and 21576040), the National Basic Research Program of China
(no. 2013CB733702).
Notes and references
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Journal of Materials Chemistry B
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Fixable probe, named Vis-A, quantify mitochondrial viscosity of living cells by fluorescence lifetime imaging