A General Strategy for the Construction of NIR-emitting Si-rhodamines and Their Application for Mitochondrial Temperature Visualization

Article abstract of DOI:10.1002/asia.202000660

Si-rhodamine (SiR) is an ideal fluorophore because it possesses bright emission in the NIR region and can be implemented flexibly in living cells. Currently, several promising approaches for synthesizing SiR are being developed. However, challenges remain

Full text of DOI:10.1002/asia.202000660

CHEMISTRY
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Accepted Article
Title: A general strategy for the construction of NIR emitting Si-
rhodamines and their application for mitochondrial temperature
visualization
Authors: Weiguo Tang, Han Gao, Jiaxin Li, Xianhui Wang, Zhikuan
Zhou, Lizhi Gai, Xin Jiang Feng, Jiangwei Tian, Hua Lu, and
Zijian Guo
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10.1002/asia.202000660
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PAPER
A general strategy for the construction of NIR emitting
Si-rhodamines and their application for mitochondrial
temperature visualization
Weiguo Tang,† [a] Han Gao, †[b] Jiaxin Li, [a] Xianhui Wang, [a] Zhikuan Zhou, [a] Lizhi
Gai, [a] Xin Jiang Feng, *[a] Jiangwei Tian,* [b] Hua Lu,* [a] and Zijian Guo*[c]
[a]
W. Tang, J. Li, X. Wang. Dr. Z. Zhou, Dr. L. Gai, Dr. X. Feng, Prof. Dr. H. Lu, Key Laboratory of Organosilicon Chemistry and
Material Technology of Ministry of Education, and Key Laboratory of Organosilicon Material of Zhejiang Province, Hangzhou
Normal University. No. 2318, Yuhangtang Road, Hangzhou 311121, P. R. China. E-mail: xjfeng@hznu.edu.cn;
hualu@hznu.edu.cn
[b]
[c]
H. Gai, Dr. J. Tian, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational
Research, China Pharmaceutical University, Nanjing 211198, China. Email: jwtian@cpu.edu.cn
Prof. Dr. Z. Guo, State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing
University, Nanjing, 210023, P. R. China. E-mail: zguo@nju.edu.cn
Supporting information for this article is given via a link at the end of the document.
Abstract: Si-rhodamine (SiR) is an ideal fluorophore because it
possesses bright emitting in the NIR region and can be implemented
flexibly in living cell. Currently, several promising approaches for
synthesizing SiR are being developed. However, challenges remain
in the construction of SiR containing functional groups for bioimaging
application. Herein, we introduce a general and simple approach by a
condensation reaction of diarylsilylether and arylaldehyde in o-
dichlorobenzene to synthesize a series of SiRs bearing various
functional substituents. These SiRs have moderate to high quantum
efficiency, tolerance to photobleaching, and high water solubility as
well as NIR emitting, and their NIR fluorescence properties can be
controlled through the photoinduced electron transfer (PET)
mechanism. Fluorescence OFF-ON switching effect is observed for
SiR 9 in the presence of acid, which is rationalized by DFT/TDDFT
calculations. Moreover, reversible stimuli response toward
temperature is achieved. Since positive charge enables mitochondrial
targeting ability and chloromethyl unit can covalently immobilize the
dyes onto the mitochondrial via click reaction between the benzyl
choride and protein sulfhydryls, SiR 8 is identified as a valuable
fluorescent marker to visualize the morphology and monitor the
temperature change of mitochondrial with high photostability.
specific targets have been developed based on red to NIR
emitters.[3] Among the fluorophores, rhodamine is often used
since it exhibits high photostability, good water solubility, and
strong fluorescence quantum yield. However, rhodamine-based
fluorophores usually show absorption and emission wavelengths
below 600 nm, which limits their application for bioimaging in living
systems.[4]
Replacement of the bridging oxygen by silicon has been shown
to dramatically red-shift their absorption wavelengths to the range
of 630-660 nm, matching the commercial red laser very well.[5]
Notably, silicon-substituent improves their resistance to photo-
bleaching, while retaining the key advantages that rhodamines
have many biological applications, such as water-solubility, pH
independency of photophysical properties, and mitochondrial
localization in cells.[6] The bathochromic shift results from the
relatively low-lying LUMO energy levels, due to σ*–π* conjuga-
tion resulting from interaction of the σ* orbital of silicon atom with
the π* system of the xanthene moiety.[6b]
The first silicon-substituted xanthene fluorophore, TMDHS(2,7-
bis(dimethylamino)-9,9-dimethyl-9-sila9H-anthracenium),
was
reported by Xiao et al in 2008.[7] Subsequently, Nagano and co-
workers developed a series of silicon-substituted rhodamines
(SiRs) with functional group at meso-position via nucleophilic
addition of aryl lithium reagents to the Si-xanthone.[8] In 2017,
Lavis and Sparr et al. developed a new way of synthesis of SiR
through reaction of the intermediate bisnucleophiles reacted with
anhydride or carboxylic acid esters electrophiles.[9] Aryllithium or
aryl Grignards are used in both synthetic methods, which are
incompatible with many functional groups, thus require the use of
complex protection strategies. Urano and Kramer et al.
synthesized the SiR by coupling the triflate of xanthone with
boroxines,[10] this method usually need multi-step synthesis.
Therefore, new strategies are urgent for construction of SiR
containing functional groups for bioimaging application.
Introduction
Fluorescent probes are the powerful and most convenient
chemical tools to investigate biological processes.[1] In particular,
red and near-infrared (NIR) fluorophores with excitation and emis-
sion in the spectral region of 600-900 nm are of high interest.
Unlike visible-light fluorophores, red and near-infrared (NIR)
fluorophores exhibit low autofluorescence interference, deep tis-
sue penetration, and minimal photodamage to biological
samples.[2] Therefore, various fluorescent probes for detecting
1
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10.1002/asia.202000660
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Scheme 1. Synthetic procedures and chemical structure of SiRs 1-12.
In other hand, mitochondria are essential organelles and play
roles in many important metabolic processes such as signaling,
cellular differentiation and apoptosis, and storage of calcium
ions.[11] Mitochondria act as the power generators of the cell and
their temperature variation may be a sign of the status of
mitochondrial energetics.[12] In some diseases, cellular
dysfunctions, originating in disordered energy metabolism, might
be revealed by abnormal changes of local temperature in
mitochondria.[13] However, monitoring in real-time the local
temperature within mitochondria is extremely challenging.[14]
Several exciting research on molecular thermometers targeting
mitochondria have been reported,[15] however, challenges remain
in the NIR dyes with high photostability, in which contain a fixable
unit to form stable covalent bonds with biomacromolecules.
We noticed that Wang et al. reported a simple synthesis for the
Si-rhodamines (SiRs) using p-toluenesulfonic acid monohydrate
catalyst without any solvent in sealed tube.[16] The authors warn
us of potential dangers (an explosion occurred when we repeated
the experiment) and the solid-state reaction is heterogeneous
reaction. Furthermore, SiRs synthesized by this method contain
both p-toluenesulfonate anion and Cl anion, leading to very
difficult purification. Herein, we reported a general approach via a
condensation reaction of diarylsilylether and arylaldehyde in o-
dichlorobenzene (o-DCB) to construct a series of Si-rhodamine
bearing various functional substituents, such as –NO2, –CH2Cl,
–N(CH3)2 and –C≡CH with moderate to high yield. We then
investigated their spectroscopic properties in which the
fluorescence properties are well controlled by the photoinduced
electron transfer (PET) mechanism, temperature-dependent
emissive behaviour and photostability. SiR 8, containing
chloromethyl unit, which can covalently immobilizes the dyes onto
the mitochondrial via “click-type” reaction between the benzyl
chloride and protein sulfhydryls, exhibits excellent properties for
visualizing the morphology and monitoring the temperature
change of mitochondrial with high photostability.[17] Our work
provides a promising strategy to design functional fluorophores
and a new platform for real time and local temperature monitoring
in mitochondria.
the solvents examined, the use of high boiling solvents is essen-
tial for this reaction, and o-dichlorobenzene was found to be op-
timal solvent. Among the several acid catalysts screened, AlCl3
was recognized as being the most effective, which afforded the
SiR 1 product in 56% isolated yield. On the basis of these results,
we examined the applicability and scope to other aryl aldehydes.
A variety of SiRs in some cases containing various functional
substituents were then synthesized in a one step process.
Accord-ingly, the present reaction provides a simple and general
proce-dure for the preparation of SiRs.
Spectroscopic Properties. Usually, rhodamine-based fluoro-
phores show absorption and emission wavelength around 530
and 550 nm, respectively. SiRs 1-12 display a narrow absorption
band in the range of 650-666 nm with high molar extinction coeffi-
cients (usually above 105 M/cm) and a weak shoulder around 610
nm (Figure 1a, and Table S3). Replacement of oxygen by silicon
results in a red shift of ca. 120 nm for the main absorption and
emission band relative to the analogous band of Rho 6G. The
main absorption band can be assigned as the main electronic
band of the S0→S1 transition. Depending on the substituent in the
meso position, the absorption maximum appears between 666
nm for SiR 5 with the strongest electron acceptor and 652 nm for
SiR 9 with the strongest electron donor (Table 1). An increase of
the substituent’s electron accepting nature results in
a
bathochromic shift of the absorption and emission bands. The
absorption energy follows a linear dependence with the electron
donor strength of R according to the Hammett constant σp (Figure
1b). Although the X-ray structure of SiR 5 reveals that the meso-
aryl group and the fluorophore are almost orthogonal for
approximately 87° in crystals (Figure S1), certain degree of
conjugation and a certain influence of the electronic nature of R
on the spectroscopic properties can be expected.[18]
The emission bands of SiRs 1-12 mirror images of their S0→S1
transition bands with moderate Stokes shifts are observed. The
absorption and emission maxima are almost independent of the
solvent polarity with a maximum solvent shift of 7 nm in CH2Cl2
and CH3CN. These features suggest that the emission originates
from the weakly polar and non-relaxed S1 state of the chromo-
phores. The full width at half-maximum height of the absorption
and emission spectra of SiRs 1-12 are narrow at approximately
below 40 nm, similar to typical BODIPY derivatives, which has the
advantages of high throughput efficiency, sensor’s sensitivity and
resolution (Table 1).
Results and Discussion
Optimization of reaction conditions and synthesis. Initially,
the effects of solvents and catalysts in the condensation reac-
tions were examined. The results are shown in Table S1. Among
2
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10.1002/asia.202000660
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Table 1. Photophysical properties of SiRs 1-12 measured in CH2Cl2 and CH3CN.
[a]
[c]
[d]
[e]
Fwhmabs
[nm]
Fwhmem
[nm]
abs
em
∆vabs-em
[cm‒1
τf
kr
knr
ε
[b]
Solvent
ФF
[nm]
[nm]
]
[ns]
[
8s-1]
[
8s-1]
M-1cm-1
10
10
1
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
655
652
653
650
666
661
665
660
666
661
666
661
664
659
658
655
652
650
663
656
659
655
663
660
29
33
42
42
38
42
31
36
29
33
30
35
30
34
29
33
39
44
28
32
32
36
31
32
669
671
666
667
677
680
677
676
679
677
678
677
675
674
674
672
667
666
676
673
673
672
677
674
37
38
38
39
44
43
38
39
37
38
37
39
36
38
35
37
36
39
35
37
38
38
38
37
319
434
299
392
244
423
267
359
287
358
266
358
245
338
361
382
345
370
290
385
316
386
312
315
0.35
0.35
0.34
0.31
0.40
0.12
0.32
0.23
0.17
0.10
0.55
0.34
0.33
0.18
0.40
0.26
<0.01
<0.01
0.27
0.25
0.41
0.34
0.80
0.65
3.09
2.50
2.68
1.77
4.07
1.45
3.06
2.31
1.57
1.04
5.31
4.64
3.33
2.78
2.40
1.95
--
1.13
1.40
1.27
1.75
0.98
0.83
1.05
1.00
1.08
0.96
1.04
0.73
0.99
0.65
1.67
1.33
--
2.10
2.60
2.46
3.90
1.47
6.07
2.22
3.33
5.29
8.65
0.85
1.42
2.01
2.95
2.50
3.80
--
132800
117200
97600
2
97100
3
97800
96800
4
109100
92900
5
98400
88000
6
109600
99100
7
111700
96400
8
113000
128000
178500
183000
125000
144100
158300
191000
145200
190000
9
--
--
--
10
11
12
2.45
1.91
3.11
2.26
5.30
4.54
1.10
1.31
1.32
1.50
1.51
1.43
2.98
3.93
1.90
2.92
0.38
0.77
[a] Stokes shift. [b] Fluorescence quantum yield using Zinc phthalocyanine (ФF = 0.30 in 1% pyridine in toluene) as the standard. The standard
errors are less than 5%. [c] fluorescence lifetime. [d] Radiation rate constant. [e] Non-radiation rate constant.
Photostability is an important parameter especially in applica-
tion of biological imaging for long-term observation. SiRs display
high stability and can persist continuous exposure to irradiation
for 40 min using a laser beam (635 nm, 200 mW/cm2). Whereas,
methylene blue (MB), with a similar absorption band with SiR,
shows significant photobleaching after irradiation (Figure 1c).
We envision that the rotation of the diethlyamino and meso-
phenyl group can lead to the decreasing of fluorescent intensity
with the increasing temperature.[19] Therefore, we explored the
tem-perature sensitivity of SiR 8 in sodium phosphate buffer
solution. As shown in Figure 1d-e, the fluorescence intensity
decreases gradually as the temperature increases from 10 to
45°C, and SiR 8 maintains its performance after going through
several temperature cycle. Accordingly, the fluorescence lifetime
reduces from 1.66 ns at 10 °C, 1.37 ns at 20 °C to 0.74 ns at 45 °C.
Moreover, fluorescence intensity is not greatly affected by pH
(Figure S2). These results show that SiR 8 could be used as a
molecular thermometer by correlating its fluorescence intensity
with temperature.
fluorescence quantum yield up to 0.80 in dichloromethane due to
the restriction of free rotation of meso-substituent resulting from
the large steric hindrance of naphthyl group. The fluorescence
decay profiles could be described with a single-exponential fit with
fluorescence life times in the 1.04–5.31 ns range (Table 1).
DFT calculations. DFT and TD-DFT calculations were carried
out to understand the spectroscopic behaviours. The substitution
of oxygen by silicon destabilize HOMO and stabilize LUMO
energy, thus a smaller energy gap is obtained and large red shift
is achieved. The low-lying LUMO energy level can be attributed
to the σ*–π* interaction between the σ* orbital of silicon atom with
the π* system of the xanthene moiety. The HOMO energy in-
crease is caused by the inductive electron-donating effect of the
dimethylsilyl group relative to oxygen.[7] (Figure S3). Usually,
quenching of the original fluorescent emission will take place
when the PET process is followed by a non-luminescent adiabatic
process returning to the ground state. The PET process may be
rationalized pictorially in terms of simple molecular orbital theory,
which was first developed by Weller[20] and has become a
prevalent tool to discuss the fluorescence on-off.[10a, 21] The meso-
aryl group and the fluorophore is almost orthogonal, the emitting
properties are well explained by PET mechanism. TD-DFT
calculations display that the main component of the allowed S0→
S1 transition of SiRs 1-8 and 10-12 and protonated 9 is the
fluorophore localized HOMO → LUMO (Table S3). Scheme 2
shows the possible pathways of fluorescence emission and
All the SiRs have moderate to high fluorescence quantum
yields (0.30-0.80), except SiR 9, which fluorescence is quenched
due to the PET from N(CH3)2 to the fluorophore. As expected,
protonation at the aniline-N atom suppresses the quenching pro-
cess and leads to a revival of fluorescence. Figure S3 demon-
strates the potential of SiR 9 as a pH light-up probe, analysis of
titration curves yields a pKs of 3.4. SiR 12 displays relatively high
3
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Rho 6G
6
8
(a)
(b)
(c)1.0
0.8
y = -0.21x+15.21
R2 = 0.87
1.2
15.4
15.3
15.2
15.1
15.0
1
6
8
10
12
MB
Abs
500
FL
2
1
9
0.8
0.4
0.0
8
0.6
11
4
5
0.4
600
/nm
700
-1.0 -0.5
0.0
p
0.5
1.0
0
8
16 24 32 40
Time /min
s
1200
(d)
(e)700
(f)
10 C
20 C
45 C
10
600
400
200
0
10 C
45 C
600
500
400
300
200
20
30
40
50
800
400
0
650
700
750
0
2
4
6
8
4
5
6
/nm
Cycle
Lifetime/ns
Figure 1. (a) Absorption (solid line) and fluorescence (dot line) spectra of Rho 6G, SiR 6 and 8 in dichloromethane. (b) Absorption maxima in
wavenumbers versus Hammett constant. (c) Comparison of photostability of methylene blue (MB), SiR 1, 6, 8, 10 and 12 in DCM at 4 min intervals
determined using a laser beam (635 nm, 200 mW/cm2) over an irradiation period of 40 min (c=10-5 M). (d) Fluorescence spectra of SiR 8 (1 μM in 0.1 M
sodium phosphate buffer, pH 7.4 containing 1% DMSO) recorded at different temperature values. (e) Cycle of temperature change and intensity at 666
nm. All data were obtained using an excitation wavelength of 610 nm. (f) Time-resolved FL decay curves of SiR 8 measured at 10, 20 and 45.
case of SiR 9, the electron transfer from aniline group to fluoro-
phore is possible due to the fact that the HOMO of the substituent
is located above the HOMO of the fluorophore. Electron transfer
takes place during the electrons reorganization to its lowest vibra-
tional state on the S1 energy surface, which prohibits the electron
on the LUMO of fluorophore from going back directly, which could
explain the low fluorescence quantum yield of SiR 9 (Scheme 2).
The lowest transition state of SiR 9 is predicted by TD-DFT using
B3LYP functional to be associated with HOMO localized on
aniline group to LUMO localized on fluorophore with small
oscillator strengths, the calculated oscillator strengths of the
corresponding emission (S1→S0) is zero, therefore, S1 is dark
state (Table S4). However, the energy levels of the molecular
orbitals change greatly when protonated, which lead to significant
changes in energy sequences of these frontier orbitals, the LUMO
orbital of the receptor is higher than the LUMO of the fluorophore
and the HOMO orbital of the receptor is lower than the HOMO of
the fluorophore, thus electron transfer from meso-N,N-
dimethylaniline group to fluorophore is forbidden (Scheme 2). The
calculated emission have large oscillator strengths thus S1→S0 is
radiative process. This explains the fluorescence enhancement
phenomena after the addition of the acid. Similar cases are
shown in Scheme 2 for SiRs 1-8 and 10-12. DFT calculations
using CAM-B3LYP/6-31+G(d) display the same trend with the
B3LYP functional (Figure S5).
Scheme 2. HOMO and LOMO energy level of the fluorophore and the aryl
moiety. These values were obtained in CH2Cl2 calculated with the
polarisable continuum model (PCM) by using the B3LYP functional with 6-
31+G(d) basis sets.
Evaluation of the mitochondria-targeting capability. Cell
viability was investigated first before applying SiR 8 in cellular
imaging. Apparently, more than 85% of the HepG2 cells were
survived under the dosage of 40 μg/mL SiR 8 (Figure S6), indi-
cating the good biocompatibility. We then evaluated the imaging
electron transfer processes. We first look into the electron
distribution and pick out the orbitals mainly localized on fluoro-
phore part to form the left column; similarly, the right column
comes from orbitals mainly distributed on the meso-aryl part. In
4
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ability of SiR 8 in live cells by confocal laser scanning microsco-
py (CLSM). Filamentous structures of the mitochondria morphol-
ogy were observed in the CLSM images (Figure S7). The fluores-
cence intensity of SiR 8 in the live cells obviously relied on the
concentration of SiR 8 and incubation time. To avoid any unnec-
essary leakage, 10 μg/mL SiR 8 and 30 min incubation time were
selected for the next experimental conditions.
Figure 2. Confocal fluorescence imaging for living cells. (a) CLSM of SiR 8 (10 μg/mL) in live HepG2 cells. (b) CLSM of Mito Tracker Green (MTG, 100
nM) in live HepG2 cells. (c) Merged image of (a), (b) and bright field image. (d) Fluorescence intensity profile of the marked line in (c). (e) Fluorescence
images of HepG2 cells stained with SiR 8 (10 μg/mL) and Rho123 (50 nM), and then treated with (right side) and without (left side) CCCP (10 μM). (f)
Normalized fluorescence intensity plots of HepG2 cells corresponding to (e). (g) Fluorescence images of SiR 8 (10 μg/mL) in HepG2 cells exposed to
external media fixed at 30, 32, 34, 36, 38 and 40 degree centigrade, respectively (top). Fluorescence images of SiR 8 (10 μg/mL) in HepG2 cells exposed
to external media at room temperature (bottom). (h) Changes in the fluorescence intensity of SiR 8 in HepG2 cells exposed to external media of different
temperature corresponding to (g). (i) Real-time monitoring of mitochondrial-temperature changes in PMA-stimulated HepG2 cells (t = 0, 5, 10, 15, 20, 25
and 30 min). (j) Changes in the fluorescence intensity of SiR 8 (10 μg/mL) with stimulation time corresponding to (i). Scale bars: 10 µm.
We then studied the cellular uptake mechanism of SiR 8. The
HepG2 cells were cultured with SiR 8 at 4 °C and 37 °C for 30
min, respectively. No significant divergence in fluorescence inten-
sity was shown in Figure S8, indicating that the cellular uptake of
SiR 8 may be non-energy-dependent mechanism, like physical
adhesion and penetration. Additionally, because the clathrin- and
caveolae-mediated endocytosis processes are the most
extensively studied endocytosis pathways for eukaryotic cells, the
effects of chemical inhibitors on the cellular internalization of SiR
8 were explored. HepG2 cells were incubated with dynasore (an
inhibitor of clathrin-mediated endocytosis) and genistein (an
inhibitor of caveolae- mediated endocytosis) to study the roles of
the endocytosis process of SiR 8. No obvious difference of the
fluorescence intensity was observed in live cells with drug
treatment compared with the control group (Figure S9). The
above results demonstrated that the physical adhesion and then
penetration was the main pathway for SiR 8 endocytosis process.
To access the mitochondria-targeting ability of SiR 8, HepG2
cells were costained with SiR 8 and commercial fluorescent track-
ers (Mito Tracker Green, Lyso Tracker Green and Hoechst 33342,
respectively). The fluorescence from SiR 8 almost completely
overlapped with that from Mito Tracker Green (Figure 2a-c). An
intensity profile of the mitochondria marked with a line in Figure
2c was used to investigate the degree of the colocation as demon-
strated in Figure 2d. The well-matched intensity peaks confirm the
excellent mitochondria-targeting ability of SiR 8 in live cells.[22] In
addition, CLSM images of live HepG2 cells stained with 10 μg/mL
SiR 8 at different sections through the zaxis were performed in
ESI, Figure S10. On the contrary, the fluorescence from SiR 8
didn’t overlap with that from Lyso Tracker Green (Figure S11) and
Hoechst 33342 (Figure S12). These results demonstrated that the
high selectivity of SiR 8 to mitochondria. By the way, the generic
mitochondrial targeting feasibility of SiR 8 was tested on other cell
lines including carcinoma cell line (human mammary cancer MCF-
7 cells) and normal cell lines (human normal liver L-02 cells and
mouse embryonic fibroblast 3T3-L1 cells). The whole cell lines
displayed clear filamentous structures (Figure S13), illustrating
great mitochondria targeting efficiency of SiR 8 in different cell
lines.
To test the immobilizing ability of SiR 8 in mitochondria, car-
bonyl cyanide 3-chlorophenylhydrazone (CCCP), a type of proto-
nophore that can decrease the mitochondrial men brane potential
(MMP),[23] was utilized to conduct a mitochondrial uncoupling
experiment. HepG2 cells were cultured with SiR 8 and Rhoda-
mine 123 at 37 °C for 30 min, respectively. Rho123 is an electro-
static-attraction based cationic mitochondrial probe which can
5
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10.1002/asia.202000660
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disengagement from mitochondria when MMP is abolished. Then
the above cells were all incubated with 10 μM CCCP for another
1 h. The SiR 8-stained mitochondria performed excellent fluores-
cence stability after CCCP incubation while the Rho123-stained
showed significant fluorescence intensity decrease (Figure 2e
and 2f). Moreover, the fluorescence intensity of Rho123 (green
channel) displayed much weaker in the presence of SiR 8 (Figure
S14), which may due to the competitive reaction of SiR 8 with
nucleophiles in mitochondria. The results demonstrated that SiR
8 exhibited stronger binding capability. Notably, the fluorescence
intensity of SiR 8-stained HepG2 cells changed negligibly while
that of Rho123 decreased obviously within 12 min (Figure S15),
illustrating the high photostability of SiR 8. Compared to com-
mercial probe Rho123, our new probe SiR 8 exhibits brilliant
imaging capability with MMP decreased, and shows stronger
binding capacity to mitochondria and excellent stability, which
could be attributed to the covalent bonding between chloromethyl
group of SiR 8 and nucleophiles in in mitochondria.
between physiological and pathological conditions or screening
for potential new mitochondrial targeting drugs.
Experimental Section
Materials and Instruments. All the starting materials were obtained from
commercial suppliers and used as received. Details are in Supporting
Information.
General synthetical method. Diarylsilylether (100 mg, 0.3 mmol),
arylaldehyde (1.5 mmol) and AlCl3 (0.3 mmol) were added in a 10 mL
Schlenk, then added 2 mL o-dichlorobenzene and heated to 140 °C for 8
hours. After cooling to room temperature, the reaction mixture was
dissolved in dichloromethane/methanol (2 mL/2 mL), to which p-chloranil
(110 mg, 0.45 mmol) was added. The reaction mixture was stirred at room
temperature 12 h, then filtrated and evaporated under reduced pressure.
The residue solid was purified by column chromatography on silica gel
(dichloromethane: methanol = 20: 1) to afford compound SiR as a dark
blue solid.
Mitochondrial temperature quantification through CLSM
imaging of living cells. SiR 8 was also utilized to monitor the
local temperature in the mitochondria of living cells by CLSM.
HepG2 cells stained with SiR 8 were incubated with culture me-
dia at different temperatures. The fluorescence intensity of SiR 8
gradually decreased as the culture temperature rose from 30 to
40 °C (Figure 2g). While the fluorescence intensity of SiR 8 at
room temperature for the same duration of time didn’t change
(Figure 2g). The results demonstrated that SiR 8 could be used
as an indicator for probing mitochondrial temperature. It was
worth noting that changes in fluorescence intensity was not
linearly related to the alteration of living cells medium temperature
(Figure 2h), which could be attributed that living cells may have
self-regulating ability to against the changes in the external
temperature. Moreover, phorbol 12-myristate-13-acetate (PMA)
as a chemical reagent to activate the protein kinase C system was
utilized to stimulate HepG2 cells, which could cause remarkable
change in mitochondrial temperature.[24] As displayed in Figure 2i
and Figure 2j, during the 30 min stimulation process, the fluores-
cence intensity of SiR 8 apparently decreased, which indicated
the increasing mitochondrial temperature of HepG2 cells.
Nevertheless, the fluorescence intensity of no PMA-treated
HepG2 cells changed slightly. All the above results confirmed that
SiR 8 could have the excellent ability to monitor the mitochondrial
temperature in living cells.
Spectroscopic measurements. Fluorescence spectra and the
fluorescence lifetimes of the samples were determined with a Horiba Jobin
Yvon Fluorolog-3 spectrofluorimeter. The goodness of the fit of the single
exponential decays were judged using the chisquared (χR2) and the
autocorrelation function C(j) values. Low residuals (χR2 < 1.2) were
consistently observed. For samples in solution, absorption and emission
measurements were carried out in 1×1 cm quartz cuvettes. The
luminescence quantum yields were measured using Zinc phthalocyanine
(ФF = 0.30 in 1% pyridine in toluene) as a reference.[25] The quantum yield
ФF as a function solvent polarity is calculated using the following equation.
푰
푨
풔풂풎풑풍풆
풔풕풅
2
Фsample=Фstd
[
풔풂풎풑풍풆][ 푨
][ 풏
]
(1)
푰
풏
풔풕풅
풔풕풅
풔풂풎풑풍풆
Where subscript sample and std denote the sample and standard,
respectively, Φ is quantum yield, I is the integrated emission intensity, A
stands for the absorbance, n is refractive index.
The rate constants of radiative (kr) and nonradiative (knr) deactivation
were calculated from the measured fluorescence quantum yield and
fluorescence lifetime (τ) according to equations (2) and (3):
kr =ФF/τ
(2)
(3)
knr = (1-ФF)/τ
Computational details. The ground state structures were optimized using
density functional theory (DFT) with B3LYP and CAM-B3LYP functional
and 6-31+G(d) basis set. The excited state related calculations were
carried out with the time dependent DFT (TD-DFT), based on the
optimized structure of the ground state. The polarizable continuum model
(PCM) is used in CH2Cl2 to model solvation effects. All these calculations
were performed with Gaussian 09.[26]
Conclusion
We have developed a general approach to prepare SiR bearing
various functional groups without protection with moderate yields.
These SiRs display moderate to high fluorescence quantum effi-
ciency, high photostability and NIR emitting, and their NIR fluo-
rescence properties could be controlled through the PET mecha-
nism. DFT and TD-DFT calculations rationalize the OFF–ON
sensing and PET mechanism. A fixable molecular thermometer,
to trace local mitochondrial temperature has been established
because positively charged probe SiR 8 has a chloromethyl group
as an anchoring unit, which enables its permanent immobilization
into mitochondria, so that it maintains sensitivity toward
temperature. Therefore, we envisage that the fixable thermometer
SiR 8 will help improve diagnostics and test dynamic monitoring
of mitochondrial temperature as a means of distinguishing
Crystallographic data. The X-ray crystallographic coordinates for the
structures reported in this article have been deposited at the Cambridge
Crystallographic Data Centre (CCDC) under deposition numbers CCDC
1985857. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Co-localization assay. The HepG2 cells were washed with PBS and
further stained with 10 µg/mL SiR 8. After incubated for 30 min, the cells
were washed with PBS twice to remove the unbound molecules.
Subsequently, the cells were stained with 100 nM MitoTracker Green
(MTG). MitoTracker Green were excited at 488 nm with an argon ion laser,
and the emission was collected from 500 to 550 nm. All images were
6
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10.1002/asia.202000660
Chemistry - An Asian Journal
FULL PAPER
analyzed by a ZEN imaging software. To evaluate whether the as-
synthesized SiR 8 can specifically target the mitochondria even if the
mitochondrial membrane potential (MMP) changed or vanished, 10 µg/mL
as-treated SiR 8 was firstly added into the HepG2 cells for 30 min at 37 °C.
50 nM Rho123 solution was injected into the other cell dish. After
incubated for 30 min, the cells were washed with PBS twice to remove the
unbound molecules. Afterwards, the media were replaced with fresh
DMEM containing CCCP (10 μM) and the cells were incubated for another
1 h, respectively; finally, the cells were washed and cell imaging was
performed as mentioned above.
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cells were heated to the set temperature (30, 32, 34, 36, 38 and 40 °C).
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Acknowledgements
We thank the National Natural Science Foundation of China (No.
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Keywords: Si-rhodamine• Dyes and Pigments•NIR emission
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10.1002/asia.202000660
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Entry for the Table of Contents
We report a general and simple approach to synthesize Si-rhodamines bearing various functional substituents and application for
monitoring the temperature change of mitochondrial.
8
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