A dual-site two-photon fluorescent probe for visualizing mitochondrial aminothiols in living cells and mouse liver tissues

Article abstract of DOI:10.1039/c6nj00330c

Thiol-containing amino acids (RSH) play a key role in maintaining the redox environment. In this work, we developed a novel dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging mitochondrial RSH in living cells and mouse liver tissues. This probe exhibited rapid response and high TP photostability towards mitochondrial RSH. In particular, probe CA-TPP was capable of using TP fluorescence to track mitochondrial RSH over a long period.

Full text of DOI:10.1039/c6nj00330c

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DOI: 10.1039/C6NJ00330C
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A dual-site two-photon fluorescent probe for visualizing
mitochondrial aminothiols in living cells and mouse liver tissues †
Fangfang Meng, a Yong Liu, a Xiaoqiang Yub*and Weiying Lina*
Received 00th January 20xx,
Accepted 00th January 20xx
Thiol-containing amino acids (RSH) play key role in maintaining the redox environment. In this work, we
developed a novel dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging mitochondrial RSH
in living cells and mouse liver tissues. This probe exhibited rapid response and high TP photostability for
mitochondrial RSH. In particular, the probe CA-TPP was capable of using TP fluorescence to track
mitochondrial RSH over a long period.
DOI: 10.1039/x0xx00000x
www.rsc.org/
with very large mitochondrial membrane potential (ΔΨm) value.11
Introduction
This value is
6 times larger than nonexcitable cell plasma
membranes. Due to this large membrane potential gradient,
mitochondria drive cationic species, such as triphenylphosphonium
salt (TPP), into the matrix.12 Their accumulation in mitochondria is
100−500-fold higher than in other parts of the living cell and tissues.
Thus, to develop a unique dual-site mitochondrial RSH functional
probe, TPP is important mitochondria sites.
Two-photon (TP) fluorescence imaging has advantages over
traditional optical imaging techniques.13 Investigations have
demonstrated that two-photon microscopy has unique merits
in imaging live specimens, and thus, various fluorescent probes
with large two-photon action absorption cross-sections (),14
including those for thiols,15 have been investigated. Recently,
mitochondria-targetable one- and two-photon fluorescent
probes have been constructed to quantify mitochondrial pH,16
Thiol-containing amino acids (RSH), such as cysteine (Cys),
homocysteine (Hcy), and glutathione (GSH), play a key role in
various biological processes; these processes include maintaining
the homeostasis of biological thiols, protein synthesis, metabolism,
post-translational modification, and detoxification of xenobiotics.1–3
RSH maintains higher-order protein structures and control redox
homeostasis through the equilibrium between thiols and
disulfides.4, 5 Detection of RSH has been widely studied in recent
years owing to their biological importance for early diagnosis of
disease, evaluation of disease progression, and the therapeutic
efficiency of potential new drugs.6–8 Consequently, clearly
separating RSH from other bioanalytes is necessary to separate
them successfully from RSH without amino groups.
In mitochondria, a primary site of oxygen consumption and the hypochlorite,17 sulfur dioxide,18,19 zinc ion,20 amidoxime,21 and
22
hydrogen sulfide
in living cells, suggesting the high
major source of reactive oxygen species (ROS), intracellular RSH
plays a key role in maintaining the redox environment to prevent or
repair oxidative damage leading to dysfunction and cell death.9
Mitochondrial RSH exists predominantly in the reduced form. For
example, a GSH: glutathione disulfate (GSSG) ratio of approximately
100:1 exists in mitochondria. An increase in the GSH/GSSG ratio is
indicative of oxidative stress conditions.10 Monitoring RSH in
mitochondria is crucial to understanding the roles of RSH in biology.
importance of mitochondria in cell biology. Mitochondrial thiol
probes have been utilized in fluorescent probe
development.23–27 More recently, several TP fluorescent
probes for the detecting intracellular RSH or mitochondria also
have been developed.28 However, only a few of them may
target mitochondrial RSH in mouse liver tissues.24 The
aforementioned concerns encouraged us to develop novel TP
fluorescent probes to detect mitochondrial RSH in living
system.
Celluar respiration occurs in mitochondria. Mitochondria are the
organelles where maintain a proton gradient across the lipid bilayer
a Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Biological Science and Technology, University of
Jinan, Jinan, Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
b Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal
Materials, Shandong University, Jinan, 250100, PR China. E-mail: yuxq@sdu.edu.cn
†
Electronic Supplementary Information (ESI) available: Experimental procedures,
characterization data, and additional spectra. See DOI: 10.1039/x0xx00000x.
Fig. 1 Design of dual-site TP fluorescent probe CA-TPP. Blue ring
indicates the TP platform. Blue balls represent the RSH reaction
sites. Red balls represent the mitochondria site.
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In this work, we developed a dual-site TP probe derived from 9- compound 6-(4-(4-bromobutoxy) styryl)-9-ethyl-9H-carbazole-
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DOI: 10.1039/C6NJ00330C
ethyl-3-styryl-9H-carbazole (CA) as the TP fluorescent platform, 3carbaldehyde (5) was prepared by the Williamson reaction of 4
aldehyde as the RSH response site, and triphenylphosphonium salt with 1,4-dibromobutane. The objective molecule CA-TPP was
(TPP) as the mitochondrial targeting moiety, 29 denoted as CA-TPP obtained by a substitution reaction between TPP and 5. The
(Fig. 1). We predicted that thiolate-triggered reaction with aldehyde products were characterized by 1H NMR, 13C NMR and HRMS in
would achieve TP detection of mitochondrial RSH in living system.
supporting information
Results and discussion
Selectivity and sensitivity
Once CA-TPP was synthesized, we evaluated the capability of CA-
TPP to detect RSH in solution. Titration of RSH with CA-TPP was
performed in a CH3OH/Tris-HCl solution (40:1, v/v). Adding RSH (Cys,
Hcy, GSH) to CA-TPP (Fig. 2) resulted in the absence of evident
changes in absorption spectrum of CA-TPP (Fig. S1 in the ESI†). The
probe CA-TPP was treated with various biologically relevant species
in pH 4.4 PBS buffer to examine its selectivity (Fig. 3b). By contrast,
CA-TPP exhibited a strong fluorescence response to RSH, however,
other bioanalytes cannot induce any fluorescence change (Fig. 3).
The results demonstrated that the probe had an excellent
selectivity to RSH over other amino acids.
Preparation of probes
Carbazole-based derivatives exhibited
a number of attractive
features,30 such as large TP absorption/action absorption cross
sections (δ/δΦ), large stoke shift, suitable excitation wavelength for
application with common lasers, and high fluorescence quantum
yield. In 2012, our group15 used the TP fluorescent probe CA to
detect RSH in living cells; TPP molecules have also been used as
imaging agents for studying mitochondrial dysfunction.31 Inspired by
the active sites of traditional TPP and CA dyes, we developed a
unique dual-site TP functional probe, CA-TPP, which is the hybrid of
traditional carbazole-class and TPP. We predicted that this dual-site
TP fluorescent probe could detect mitochondrial RSH in living
system.
Fig. 2 Selectivity of CA-TPP in mixtures (CA-TPP, CA-TPP+Hcy, CA-
TPP+Cys, CA-TPP+GSH). [Hcy] = [GSH] = [Cys] = 1 × 10−3 M. [CA-TPP]:
50 µM.
Scheme.1 Synthesis of the Probe CA-TPP. a) Dimethylformamide
(DMF), CH3Cl and POCl3 55oC; b) N-bromosuccinimide (NBS), CH3Cl
/AcOH (1:1, v/v), 25oC; c) Pd(AcO)2/Tri-o-tolylphosphine, DMF, 4 -
o
vinylphenyl acetate, Argon, 120 C ; d) NaOH, EtOH, 25oC; e) NaH,
DMF, 25oC; f) Triphenylphosphine, p-xylene, 130 oC.
As shown in Scheme 1, the compound 9-ethyl-9H-carbazole-3-
carbaldehyde (1) was prepared in one step by the Vilsmeier
reaction of 9-ethyl-9H-carbazole with phosporus oxychoride. The
synthesis of the compound 6-bromo-9-ethyl-9H-carbazole-3-
carbaldehyde (2) was based on a reported procedure.15 The
Fig. 3 Selectivity of CA-TPP in solution. Fluorescence intensity at
410 nm of CA-TPP in solutions containing different amino acids or
analogues. [CA-TPP]: 50 μM, Concentration of analytes are
1 × 10−3 M. (D-(-)-glutamic acid (D-Glu), serine (Ser), valerian (Val),
threonine (Thr), alanine (Ala), isoleucine (Ile), phenylalanine (Phe),
asparagine (Asn), glycine (Gly), tyrosine (Tyr), methionine (Met), D-
phenylalanine (Dpa), dithiothreitol (DTT) and vitamin C (VC), proline
(Pro), leucine (Leu), tryptophan (Trp), asparagine (Asn)).
compound
4-(2-(9-ethyl-6-formyl-9H-carbazol-3-yl)vinyl)phenyl
acetate (3) was made by a Heck coupling reaction between 2 with
4-vinylphenyl acetate. The compound6-(4-hydroxystyryl)-9-ethyl-
9H-carbazole-3-carbaldehyde (4) was made in two steps.
Compound 3 was treated with sodium hydroxide in ethanol, which
was then reacted with hydrochloric acid to give compound 4. The
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To shed light on the optical properties and robust chemical stability
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of CA-TPP dyes, DFT calculations withDOthI:e10.B1033L9Y/PC6NeJx0ch03a3n0gCe
functional employing 6-31G* basis sets using a suite of Gaussian 09
programs were conducted.38 Natural bond orbital (NBO) analysis
was performed by using the NBO Version 5.9 implemented in
Gaussian 09 programs. As representative cases, CA-TPP (carbazole
and benzene forms) was studied with Gaussian 09 programs. Fig. 5
illustrated the optimized structures of CA-TPP. The C-C (N or O)
bond lengths (in pm) of CA-TPP determined by DFT calculations are
displayed in Table S2 (ESI†). The HOMO/LUMO molecular orbital
plots of CA-TPP are exhibited in Fig. 6.
For simplicity, CA-TPP structure was divided into two parts (parts
A and B). As shown Fig. 5, part A was composed of a carbazole
moiety, and part B contains a benzene moiety. Part B was highly
twisted and almost perpendicular to part A, with a dihedral angle of
79.88°. The C12–C16 and C17–C18 bond lengths were 147.5.5 and
148.0 pm, respectively, which were close to the typical C–C single
bond (154 pm) (Table S2 in the ESI†). These data showed that parts
A and B were almost electronically decoupled. That was, they were
orthogonal and had virtually no electronic conjugation.
Fig. 4 (A) Fluorescence intensity change at 410 nm in varied equiv.
of Cys. (B) Change curve of the fluorescence intensity. (C) Plot of
fluorescence intensity as a function of concentration of Cys. (D)
Change curve of response time of the probe CA-TPP and Cys. [CA-
TPP]: 50 µM.
In addition, CA-TPP was pH insensitive at biological pH values (Fig.
S2 in the ESI†). These results demonstrated that CA-TPP may serve
as
a fluorescent probe to detect intracellular RSH without
interference from other biologically relevant analytes and pH. In
fluorescence titration experiments, when the probe was excited at
365 nm, the fluorescence intensity evidently increased at 410 nm
with the addition of Cys (from 0 equiv to 30 equiv) (Fig. 4A). And
fluorescence intensity changes of CA-TPP were shown in Fig. 4B. It
showed 10-fold fluorescence intensity enhancement. Based on our
previous work, intracellular Cys can be detected when the
corresponding fluorescence intensity is more than three times as
strong as that of the probe alone.32, 33 Sensitivity is an important
criterion for practical application of probe.34–37 The detection limit
of Cys was calculated to be 0.2 µM (Fig. 4C), indicating that the
probe was highly sensitive to Cys. In addition, as shown in Fig. 4D,
we further investigated the response time of CA-TPP to Cys. The
result demonstrates that the probe had high stability in 10 min.
Prior to the imaging studies in the living tissues, it is necessary to
investigate the TP properties of the probe CA-TPP in the absence or
presence of RSH. In TP spectra, the probe CA-TPP showed strong
fluorescence response in the prescence of RSH (Fig. S3 in the ESI†).
In TP titration expriment, when the probe was excited at 730 nm,
the fluorescence intensity evidently increased at 410 nm with the
addition of Cys (Figs. S4A-B in the ESI†) . It was consisitance with
one-photon titration. And the detection limit was 10 μM at 730 nm
excitation (Fig. S4C in the ESI†). As shown in Table S1 (ESI†), the TP
absorption cross sections (δ) of the probe was negligible at 760–780
nm. However, in the presence of Cys, Hcy and GSH, the δ of the
probe was 1-10.5 GM at 760 -780 nm. These results suggested that
the probe exhibited good TP properties in vitro, and it should be
promising for monitoring RSH in living tissues.
Fig. 5 DFT optimized structure of CA-TPP. In the ball-and-stick
representation, carbon, nitrogen, and oxygen atoms are colored in
gray, blue, and red, respectively. H atoms were omitted for clarity.
Fig. 6 DFT-optimized structures and molecular orbital plots (LUMO
and HOMO) of CA-TPP based on the optimized ground-state
geometry (S0).
Time-dependent DFT (TDDFT) calculations indicated that CA-TPP
possesseed a strong absorption band at long wavelengths (f
[oscillator strength] = 0.0398) attributed to the S1←S0 transition
and a weak absorption band at short wavelengths (f = 0.0011)
assigned to the S3←S0 transition. Thus, the results of TDDFT
calculations agreed well with the experimentally obtained
absorption spectra of CA-TPP. The lowest unoccupied and highest
occupied molecular orbitals had slight correlation, and the
fluorescent intensity of the compound CA-TPP was very weak in the
solid state. Fig. S5 (ESI†) showed the solvatochromic study of CA-
TPP. The fluorescence intensities were high in organic solvents and
Density functional theory (DFT) calculations and photophysical
properties
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the image of the commercial mitochondria probe MTR (Fig. S10 in
DOI: 10.1039/C6NJ00330C
the ESI†). In addition, Changes in the mitochondrial membrane
extremely low in Tris-HCl buffer and aqueous solution. The result
demonstrated that the probe may exist in an aggregated state. Thus,
the results of the experiment were in good agreement with the
TDDFT calculations of the compound CA-TPP.
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potential (ΔΨm) value cause changes in the tolerance of the probe.
Next, we study the influence of ΔΨm by the cell imaging. The probe
MTR and CA-TPP incubated SiHa cells treated by 5 μM CCCP.
Compared with MTR, the sensitivity of CA-TPP to mitochondria is
perfectly retained in the CCCP-treated cells (Fig. S11 in the ESI†).
The results showed that intracellular RSH may also play an
important role in retaining the sensitivity of CA-TPP in CCCP-treated
SiHa cells.
With the probe in hand, we did the thiol elimination experiment.
When A549 were pretreated with the thio-blocking reagent NEM
(Fig. 8A and B), a well-known thiol-blocking agent, the fluorescence
signals from CA-TPP decreased by ~64 % (Fig. 8C). These findings
demonstrated that CA-TPP was capable of detecting mitochondrial
RSH in living cells.
Mechanism
A possible mechanistic pathway to detect RSH was proposed in
Scheme 2. This probe was initially constructed by linking TPP and an
aldehyde moiety separately to the 3,6 position of a 9-ethylcarbazole
core, producing a D––A electronic structure. Upon the reaction of
CA-TPP with RSH, the electronic structure was transformed from D–
–A to D––D owing to the transition of aldehyde to a thiazole ring,
and the fluorescent intensity of the probe exhibited a notable
enhancement. That is, the fluorescence was turned off from the ICT
effect. Based on our experimental results, the formation of
thiazolidine between CA-TPP and Cys can be explained by the
proton nuclear magnetic resonance method (Fig. S6 in the ESI†). In
deuterated methanol, the aldehyde proton peaks of CA-TPP at
10.08 ppm disappear and two new peaks at 5.55 and 4.29 ppm
become apparent after the addition of Cys.
Scheme. 2 Mechanism of RSH sensing by CA-TPP
Fig. 7 (A) Bright-field image. Confocal fluorescence images of A549
cells stained with (B) CA-TPP (5 μM) collected between 475-500 nm
upon excitation at 405 nm and (C) MTR (2.5 μM) collected
between >561 nm upon excitation at 561 nm. (D) Merged image of
B and C. (E) Intensity profile of the region of interest (indicated by
the white color line in (B) and (C) across the A549 cells. Red line for
c and green line for b. (f) Correlation plot of CA-TPP and MTR
intensities. Scale bar = 20 μm.
High-resolution mass spectrometry (HRMS) characterization
provides further evidence. In the obtained HRMS spectrum, isolated
CA-TPP + Cys thiazolidine displayed strong peaks (Fig. S7 in the ESI†).
The molecular ion peaks at m/z = 761.2294 and 381.2215
corresponding to [M+Cys]+ and [M+Cys+H]2+, respectively, are
typical of CA-TPP+Cys thiazolidine. The fragment peak at
m/z = 658.4033 [M]+ (calculated value 658.2900) corresponding to
CA-TPP indicates that CA-TPP has incomplete reaction with Cys. we
have done mass spectrum titration experiment to study
mechanistic pathway. Mainly reason may be reaction rate of CA-
TPP and Cys lead to the probe CA-TPP had incomplete reaction with
Cys. Mass spectral analysis has given good evidence for thiazolidine
formation. The result also confirms that thiazolidine is formed by
the interaction of aldehyde with Cys.
Colocalization and thiol elimination experiment
To prove the probe can detect mitochondrial RSH in living cells, we
did colocalization and thiols elimination experiments. First, the
cytotoxicity of CA-TPP was evaluated using MTT assay. The results
indicated that the probe generally presented low toxicity for living
cells (Fig. S8 in the ESI†). Second, to confirm CA-TPP can specifically
stain the mitochondria, colocalization experiment was carried out
Fig. 8 (A) Confocal fluorescence images of A549 incubated with 5
µM CA-TPP for 0.5 h at 37 °C. (B) A549 treated with 5 mM N-
ethylmaleimide for 0.5 h at 37 °C and then incubated with 5 µM CA-
TPP for 0.5 h at 37 °C. (C) The fluorescence intensity before and
after the cells were treated. Images were acquired using 405 nm
excitation and fluorescence emission windows of 425-475 nm.
Scale bar: 50 μm.
with CA-TPP and MTR (structure shown in Fig. S9 in the ESI†),39-41
.
The pearson’s colocalization coefficient of CA-TPP and MTR was
0.90, indicating that CA-TPP was predominantly located in the
mitochondria. In wide-field image, the results of colocalization
experiment showed that the fluorescence image merged well with
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One- and two-photon photostability
ARTICLE
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DOI: 10.1039/C6NJ00330C
Photostability was an important criterion for developing fluorescent
probes, because under the high-intensity illumination conditions
used for fluorescence microscopy, the irreversible destruction of
the excited fluorophore often becomes the factor limiting
fluorescence detectability. Continuous scanning by mercury lamp in
the focal plane induced was used to quantitatively investigate the
photostability of CA-TPP. Within 300 s, the fluorescence signals of
CA-TPP decreased slightly (Fig. S12 in the ESI†). We used TP
confocal microscope microscope to image A549 cells. within 360 s,
TP fluorescence signals of CA-TPP decreased slightly increase ~ 5 %
at 770 nm (Fig. 9). The results indicated that CA-TPP displayed good
photostability. Moreover, the probe CA-TPP can track
mitochondrial RSH in living cells over a long period.
Fig. 10 TP tissue 3D images were acquired at 770 nm, with
fluorescence emission windows of 475–500 nm. Compound
concentration: 30 µM
Conclusions
In summary, we designed and synthesized a dual-site two-photon
fluorescent probe CA-TPP for detecting RSH in living cells and
mouse liver tissues. This probe exhibited rapid response, good two-
photon properties, highly sensitive and good photostability to RSH
in vitro. Moreover, the probe CA-TPP was capable of tracking
mitochondrial RSH over a long period under two-photon excitation.
This dual-site probe was expected to have broader applications for
developing various dual-site fluorescent probes to sensing other
biological analytes.
Experimental
Materials
All chemicals used are of analytical grade, and carbazole, sodium
hydride, 1, 4-dibromobutane and N-bromosuccinimide (NBS) were
purchased from Sinopharm Chemical Reagent Co., MitoTracker Red,
Ltd (Shanghai, China), Palladium (II) acetate and tri-o-
tolylphosphine were purchased from J&K Chemical (Beijing, China).
Tris(hydroxymethyl)aminomethane (Tris) and phosphate buffer
saline (PBS) were purchased from Seikagaku Corporation (Japan).
The solvents used in the spectral measurement are of
chromatographic grade. Spectroscopic measurements of were
performed in methanol /Tris-HCl buffer solution (Tris: 10 mM, KCl:
100 mM) with pH 7.2. Thin layer chromatography (TLC) analyses
were performed on silica gel plates and column chromatography
was conducted over silica gel (mesh 200–300), both of which were
obtained from Qingdao Ocean Chemicals.
Fig. 9 Fluorescent images of living A549 incubated with CA-TPP.
Excitation wavelengths: 770 nm; Power: 60 µW. [CA-TPP]: 5 µM;
Detection wavelength: 425-475 nm.
Tissue imaging
The TP imaging and in situ emission profiles of the probe incubated
with the cells are displayed in Fig. S13 (ESI†). The cells exhibited
strong TP fluorescence in the blue channels, in agreement with the
results of fluorescent spectra of probe in vitro and OP imaging.
These results suggested that the probe exhibited good TP
properties in vitro and in vivo. These results showed that the probe
CA-TPP can monitor mitochondrial RSH in living tissues. TP
fluorescence microscopy is suitable for 3D fluorescence imaging in
thick living samples. As no 3D fluorescence imaging of
mitochondrial RSH in living tissues has been reported, we were
interested in exploiting the capability of CA-TPP in this regard. Slices
of liver were prepared and subjected to TP fluorescence
microscopic analysis. We used 770 nm excitation to image RSH in
deep tissues. As shown in Fig. 10, when tissue slices were incubated
with 30 µM CA-TPP for 1 h, significant fluorescence was observed
up to 70 µm. TP tissue 3D images were acquired using 770 nm
excitation and fluorescence emission windows of 475–500 nm.
These data indicated that the CA-TPP can detect mitochondrial RSH
at 70 µm depth in living tissues.
Method
Nuclear magnetic resonance spectra (1H and 13C) were obtained on
a Bruker Avanace 300/400 spectrometer. The high resolution mass
spectrometry (HRMS) spectra were recorded on Agilent
Technologies 6510 Q-TOF LC/MS or ThermoFisher LCQ Fleet. The
elemental analyses were performed on a vario EI III instrument. The
ultraviolet (UV)-visible-near-Infra-red (IR) absorption spectra of
dilute solutions were recorded on a cary 50 spectrophotometer
using a quartz cuvette having 1 cm path length. Wide-field
fluorescence microscopy images were acquired with an Olympus
IX71 inverted microscope coupling with a: Charge-coupled Device
(CCD) and display controller software. The fluorescence of CA-TPP
was excited and collected through mercury lamp. Relative
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fluorescence intensity of the intracellular cells was obtained with (1:1, v/v) and the resulting solution was allowed to reVaicewt fAortric1le5Ohnlianet
DOI: 10.1039/C6NJ00330C
NiKON Microscopy and Carl Zeiss Microscopy Cell Observer.
room temperature. The mixture was poured into water (500 mL)
and then extracted by CH2Cl2. The organic layer was washed by
saturated NaCl solution for several times before being dried with
MgSO4. After CH2Cl2 was evaporated, the crude product was
purified by column chromatography with ethyl acetate/petroleum
ether (10:1, v/v) as eluent, and finally a white solid was obtained for
Quantum yields
The fluorescence quantum yields can be calculated by means of
equation (1):
1
2 with a yield of 81%. H NMR (300 MHz, CDCl3): δ (ppm) 10.10 (s,

2 


r (r ) ns Fs
1H), 8.56 (d, J = 1.2 Hz, 1H), 8.27 (d, J = 1.8 Hz, 1H), 8.05 (dd, J1 = 8.6
Hz, J2 = 1.5 Hz, 1H), 7.62 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H), 7.49 (d, J
= 8.4 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 4.4 (q, J = 7.3 Hz, 2H), 1.47 (t, J
= 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3), δ (ppm): 191.53, 143.73,
139.33, 129.46, 128.93, 127.54, 124.80, 124.44, 123.61, 122.10,
113.20, 110.60, 109.05, 38.14, 13.80. HRMS (m/z): [M+H]+ calcd for
C15H12BrNO: 302.0100; found, 302.01649.









s  r
2

s (s )
Fr
nr


(1)
Where the subscripts s and r refer to the sample and the reference,
respectively. Ф is quantum yield, F is the integrated emission
intensity, A stands for the absorbance, and n is refractive index.
Two-photon absorption (TPA) cross sections
Synthesis
of
(E)-4-(2-(9-ethyl-6-formyl-9H-carbazol-3
yl)vinyl)phenyl acetate (3): To a solution of 2 (1.40 g, 1 equiv),
palladium acetate (0.058 g, 0.05 equiv) and tri-o-tolylphosphine
(1.64 g, 0.1 equiv) in dry DMF (45 mL) were added 4-vinylphenyl
acetate (1.20 mL, 2.3 equiv) and triethylamine (5.00 mL, 5.40 mmol)
under argon. The mixture was then immersed in a preheated oil
bath (120 °C) for 2 days. The reaction was monitored by TLC using
CH2Cl2 as eluent. The reaction was stopped after all 2 was
consumed. The resulting mixture was diluted with methylene
chloride and filtered, which was carefully washed with CH2Cl2. DMF
was removed by water extraction and the resulting solution was
dried over sodium sulfate, filtrated and concentrated under
reduced pressure. The crude product was purified by flash
chromatography using hexane/CH2Cl2 (1:1, v/v) as eluent to give
0.63 g (36 %) of 3 as pale yellow solid. 1H NMR (300 MHz, DMSO-d6),
 (ppm): 10.08 (s, 1H), 8.80 (d, J = 1.2 Hz, 1H), 8.57 (s, 1H), 8.02 (dd,
J = 8.6, 1.5 Hz, 1H), 7.79-7.83 (m, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.64-
7.69 (m, 2H), 7.32-7.45 (m, 2H), 7.14-7.18 (m, 2H), 4.53 (m, 2H),
2.29 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ
(ppm): 191.75, 169.12, 149.49, 143.43, 140.02, 135.11, 129.46,
129.09, 128.36, 126.97, 126.86, 125.79, 125.47, 123.85, 122.74,
122.34, 122.03, 118.67, 110.07, 109.73, 37.44, 20.78, 13.68. HRMS
(m/z): M+ calcd for C25H21BrNO3: 383.1500; found, 384.1597.
Two-photon absorption (TPA) cross sections were measured using
the two-photon induced fluorescence method, and the cross
section can be calculated by means of equation (2):
r cr nr F
s
s r
s cs ns F
r
(2)
Where the subscripts s and r refer to the sample and the reference,
respectively. The terms c and n are the concentration and refractive
index of the solution, respectively. F is TP excited fluorescence
integral intensity. Φ is the fluorescence quantum yield. r is the TP
absorbance cross-section of fluorescein in methanol ( = 28.5 GM)
at 760 nm.
Synthesis and characterization
Synthesis of 3-formyl-9-ethyl-carbazole (1): DMF (1.86 mL)
was added into a drying round-bottom flask and the system
was cooled to 0 °C. A solution of CHCl3 (15 mL) containing
compound 9-ethyl-9H-carbazole (4.87 g, 25 mmol) was then
added following the addition of redistilled POCl3 (2.3 mL). Then,
the solution mixture was refluxed for 16 h. After most of
CHC13 was removed, the residue was poured into ice water
and pH was then adjusted to 7–8 by NaHCO3. The water layer
was extracted by CH2Cl2 and the organic layer was washed by
water for several times before dried with MgSO4. After CH2Cl2
was removed, the crude product was purified by column
chromatography with ethyl acetate/petroleum ether (10:1, v/v)
Synthesis
of
(E)-6-(4-hydroxystyryl)-9-ethyl-9H-carbazole-3-
carbaldehyde (4): The compound 3 (0.69 g, 1.80 mmol) and 60 mL
of sodium hydroxide (1M) were stirred in ethanol (60 mL) at 25 °C.
After completion of the reaction (TLC), 65 mL of hydrochloric acid (1
M) were added. The precipitate was filtered to give the phenol,
which was purified by column chromatography using CH2Cl2 as
eluent to give a yellow solid 4 (yield: 93 %). 1H NMR (300 MHz,
DMSO-d6), δ (ppm): 10.07 (s, 1H), 9.53 (s, 1H), 8.78 (d, J = 1.2 Hz,
1H), 8.50 (s, 1H), 8.00 (dd, J = 8.6, 1.5 Hz, 1H), 7.67-7.80 (m, 3H),
7.45 (d, J = 8.7 Hz, 2H), 7.16-7.28 (m, 2H), 6.80 (d, J = 8.4 Hz, 2H),
4.51 (m, 2H), 1.36 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6), δ
(ppm): 192.76, 157.96, 144.41, 140.66, 131.16, 129.51, 129.31,
128.49, 127.77, 127.60, 126.67, 126.44, 124.94, 123.79, 123.39,
119.13, 116.56, 111.01, 110.68, 14.72. HRMS (m/z): M+calcd for
C23H19NO2: 342.1500; found, 342.1481.
as eluent, and finally the primrose solid was obtained for
1
1
(yield: 87%). H NMR (300 MHz, CDCl3): δ (ppm) 10.10 (s, 1H),
8.61 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.01 (dd, J1 = 8.4 Hz,
J2=1.5 Hz, 1H), 7.47–7.57 (m, 3H), 7.26–7.35 (m, 1H), 4.40 (q, J
= 7.2 Hz, 2H), 1.47 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3),
δ (ppm): 191.75, 143.54, 140.66, 128.50, 127.17, 126.71,
123.98, 123.13, 123.05, 120.78, 120.29, 109.13, 108.66, 37.90,
13.80. HRMS (m/z): M+ calcd for C15H13NO: 224.1000; found,
224.1138.
Synthesis of 3-formyl-6-bromo-9-ethyl-carbazole (2): Under the
protection of argon, compound 1 (2.20 g, 9.85 mmol) and NBS (1.90
g, 10.67 mmol) were dissolved in the 60 mL of chloroform/AcOH
Synthesis of (E)-6-(4-(4-bromobutoxy)styryl)-9-ethyl-9H-carbazole-
3-carbaldehyde (5): Sodium hydride (1.75 g, 4.40 mmol, 60 %) was
6 | J. Name., 2012, 00, 1-3
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added to a dry Schlenk tube containing a magnetic stirrer and then parameter hybrid functional with the LYP correlation functional
View Article Online
DOI: 10.1039/C6NJ00330C
dried in vacuo. DMF (4 mL) was then added, and the suspension (B3LYP) and 6-31G(d, p) basis set.
was stirred for 10 min at room temperature. A solution of 4 (4.90
Cell culture and staining
mg, 1.45 mmol) in DMF (10 mL) was added dropwise to the reaction
vessel causing bubbling and the appearance of a yellow/orange
precipitate. After 3 h stirring at room temperature, the reaction
mixture had become an orange suspension. A solution of 1, 4-
dibromobutane (0.88 mL, 7.30 mmol) in DMF (4 mL) was then
added dropwise to the ice-cooled reaction mixture, which was
subsequently allowed to warm to room temperature overnight to
give a clear yellow solution. Distilled water (20 mL) was then added,
and the mixture was partitioned with dichloromethane. The organic
layers were dried (MgSO4) and evaporated to dryness in vacuo. The
residue was purified by column chromatography using
CH2Cl2/petroleum ether (2:1, v/v) as eluent to give a pale yellow
solid (0.46 g, 67.2 %). 1H NMR (400 MHz, DMSO-d6), δ (ppm): 10.07
(s, 1H), 8.78 (d, J = 0.92 Hz, 1H), 8.53 (s, 1H), 8.02 (dd, J = 8.6, 1.3 Hz,
1H), 7.76-7.80 (m, 3H), 7.55 (d, J = 8.7 Hz, 2H), 7.28 (s, 2H), 6.97 (d, J
= 8.7 Hz, 2H), 4.52 (q, J = 7.2 Hz, 2H), 4.04 (t, J = 6.2 Hz, 2H), 3.63 (t,
HeLa, SiHa and A549 cells were grown in H-DMEM (Dulbecco’s
Modified Eagle’s Medium, High Glucose) supplemented with 10%
FBS (Fetal Bovine Serum) in a 5 % CO2 incubator at 37 °C. Cells
(1×105 / mL) were placed on glass coverslips and allowed to adhere
for 24 h. For living cells imaging experiment of CA-TPP, cells were
incubated with 5 μM CA-TPP in DMEM for 30 min at 37 °C. After
rinsing with PBS twice, cells were imaged immediately. For N-
ethylmaleimide (NEM) treated experiment, SiHa cells were
pretreated with a PBS solution of 5 mM NEM for 30 min in a 5 %
CO2 incubator at 37 °C, and then incubated with 5 μM CA-TPP in
PBS for 30 min at 37 °C. Fluorescence imaging was then carried out
after washing cells with the PBS buffer.
A549 and SiHa cells were grown overnight on a 35 mm petri dish
with a cover slip or a plasma-treated 25 mm round cover slip
mounted to the bottom of a 35 mm petri dish with an observation
window. The living cells were stained with 5 μM of CA-TPP for 30
min (by adding 5 μL of a 1 mM stock solution of CA-TPP in DMSO to
1 mL culture medium) or 250 nM MTR for 20 min (by adding 3 μL of
a 200 μM stock solution of MTR in DMSO to 1 mL culture medium).
For the dye entering into cells process test, the cells were imaged
by wide-field microscope. CA-TPP was excited at 330-385 nm and
the fluorescence was collected at >410 nm. MTR was excited at 561
nm and fluorescence was collected at >561 nm.
J = 6.7 Hz, 2H), 1.99 (m, 2H), 1.86 (m, 2H), 1.36 (t, J = 7.0 Hz, 3H). 13
C
NMR (100 MHz, DMSO-d6), δ (ppm): 192.29, 158.47, 143.97, 140.32,
130.64, 130.51, 128.88, 127.92, 127.36, 127.21, 126.71, 126.10,
124.39, 123.31, 122.93, 118.80, 115.26, 110.58, 110.26, 67.14,
38.00, 35.30, 29.59, 27.90, 14.25. HRMS (m/z): M+ calcd for
C27H26BrNO2: 476.1200; found, 476.1158.
Synthesis of CA-TPP: The solution of triphenylphosphine (0.52 g, 4.0
mmol) and 5 (0.24 g, 0.5 mmol) in p-xylene was heated at reflux
under argon with stirring for 48 h. Petroleum ether was then added
and a precipitate appeared and filtered. The residue was further
purified by silica gel column chromatography from dichloromethane
and eluting with methanol/dichloromethane (1:18, v/v). The
solvents were removed in vacuo to give CA-TPP as a yellow solid
(0.26 g, 70 %). IR (cm-1): 1675 (υC=O), 1238 (ΥC-O-C). 1H NMR (300 MHz,
DMSO-d6), δ (ppm): 10.08 (s, 1H), 8.78 (d, J = 1.2 Hz, 1H), 8.53 (s,
1H), 8.02 (dd, J = 8.4, 1.5 Hz, 1H), 7.91-7.94 (m, 3H), 7.69-7.91 (m,
15H), 7.55 (d, J = 8.7 Hz, 2H), 7.28 (s, 2H), 6.92 (d, J = 8.7 Hz, 2H),
4.52 (q, J = 6.9 Hz, 2H), 4.08 (t, J = 6.0 Hz, 2H), 3.68 (m, 2H), 1.94 (m,
2H), 1.74 (m, 2H), 1.36 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-
d6), δ (ppm): 192.80, 158.79, 144.45, 140.81, 135.90, 134.62, 134.49,
131.29, 131.13, 130.94, 129.35, 128.37, 127.94, 127.74, 127.14,
126.57, 124.79, 123.79, 123.39, 120.02, 119.30, 118.89, 115.77,
111.08, 110.75, 66.98, 30.25, 30.03, 21.06, 20.39, 19.49, 14.74.
HRMS (m/z): [M-Br]+. Calcd for C45H41NO2P, 658.3000; found,
658.4094.
Cell viability evaluated by MTT assay
Viability of the cells was assayed using cell proliferation Kit I with
the absorbance of 492 nm being detected using a Perkin-Elmer
Victor plate reader. Five thousand cells were seeded per well in a
96-well plate. After overnight culture, various concentrations of CA-
TPP were added into the 96-well plate and then 10 μL MTT (5
mg/mL in PBS) was added. Subsequently, the culture medium was
removed, and 100 μL DMSO was added into the dishes to dissolve
the formazan crystal product. The optical density readings at 492
nm were taken using a plate reader.
Cell imaging with carbonyl cyanide m-chlorophenylhydrazone
(CCCP) treatment
SiHa cells were grown overnight on a 35 mm petri dish with a cover
slip. The cells were incubation with 10 μM CCCP (by adding 1 μL of a
200 mM stock solution of CCCP in DMSO to 2 mL culture medium)
for 30 min. The CCCP treated cells were then stained by 5 μM of CA-
TPP for 30 min or 250 nM MTR (by adding 2.5 μL of a 1 mM stock
solution of MTR in DMSO to 1 mL culture medium) for 20 min.
Preparation of CA-TPP+Cys: A mixture of CA-TPP (0.18 g, 0.25
mmol) and Cys (0.12g, 1 mmol) in ethanol (60 mL) was stirred at
room temperature for three days. The ethanol was concentrated in
vacuo , and then the solid was collected, washed with ethanol and
dried to afford the thiazolidine.
Acknowledgements
For financial support, this work was financially supported by the
NSFC (51503077, 21172063, 21472067), Taishan Scholar
Foundation (TS 201511041), Natural Science Foundation of
Shandong province (ZR2015PE001) and the start up fund of
University of Jinan and doctor start up fund (160082102) of
Density functional theory (DFT) calculations
Density functional theory (DFT) calculations were carried out with
the GAUSSIAN 09 program package. All the calculations were
performed on systems in the gas phase using the Becke’s three-
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Journal Name
29 C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam and B.
University of Jinan. In addition, we thank Shandong University
2011JC006, Open Project of State Key Laboratory for
Supramolecular Structure and Materials (SKLSSM201207)，and the
NSFC (51303097).
View Article Online
Z. Tang, J. Am. Chem. Soc., 2013, 135, 62.
30 X. Liu, Y. M. Sun, Y. H. Zhang, F. Miao, DGO. IC: .10W.10a3n9g/,CH6N. JS0. 0Z3h3a0oC,
X. Q. Yu, H. Liu and W. Y. Wong, Org. Biomol. Chem., 2011, 9,
3615.
31 S. E. Abu-Gosh, N. Kolvazon, B. Tirosh, I. Ringel and E. Yavin,
Mol. Pharm., 2009, 6, 1138.
32 L. Yuan, W Lin, Y. Yang, Chem. Commun., 2011, 47, 62757.
33 D. S. Y. Yeo, R. Srinivasan, M. Uttamchandani, G. Y. J. Chen, Q.
Zhu and S. Q. Yao, Chem. Commun., 2003, 23, 2870.
34 M. Yu, X. Wu, B. Lin, J. Han, L. Yang and S. Han, Anal. Chem.,
DOI: 10.1021/acs.analchem.5b00847.
35 Q. -S. Lu, L. Dong, J. Zhang, J. Li, L. Jiang, Y. Huang, S. Qin, C. -
W. Hu and X. -Q. Yu, Org. Lett., 2009, 11, 669.
Notes and references
1
2
3
4
5
C. Hwang, A. J. Sinskeyand and H. F. Lodish, Science., 1992,
257, 1496.
Y. Yuan, R. T. K. Kwok, G. Feng, J. Liang, J. Geng, B. Z. Tang, B.
Liu, Chem. Commun., 2014, 50, 295.
H. Lv, X.-F. Yang, Y. Zhong, Y. Guo, Z. Li and H. Li, Anal. Chem.,
2014, 86, 1800.
R. Kizek, J. Vacek, L. Trnkova and F. Jelen, Bioelectrochem.,
2004, 63, 19.
36 Y. Peng, Y. –M. Dong, M. Dong and Y. -W. Wang, J. Org.
Chem., 2012, 77, 907.
37 Q. Wang, L. Zhou, L. Qiu, D. Lu, Y. Wua and X. –B. Zhang
Analyst., 140, 5563.
,
H. S. Jung, K. C. Ko, G.-H. Kim, A.-R. Lee, Y.-C. Na, C. Kang, J.Y.
Lee, J. S. Kim, Org. Lett., 2011, 13, 1498.
38 M. J. Frisch, GAUSSIAN 09 (Revision A.02), 2009, Gaussian,
Inc.: Pittsburgh, PA.
39 H. S. Jung, J. Han, J. –H. Lee, J. H Lee, J. –M. Choi, H. –S.
Kweon, J. H. Han, J. –H. Kim, K. M. Byun, J. H. Jung, C. Kang,
and J. S. Kim, J. Am. Chem. Soc., 2015, 137, 3017.
40 R. Kumar, J. Han, H. –J. Lim, W. X. Ren, J. –Y. Lim, J. –H. Kim,
and J. S. Kim, J. Am. Chem. Soc., 2014, 136, 17836.
41 J. -T. Hou, K. Li, J. Yang, K. -K. Yu, Y. -X. Liao, Y. -Z. Ran, Y. -H.
6
7
S. R. Lentz and W. G. Haynes, J. Med., 2004, 71, 729.
S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D’Agostino and P. W. F. Wilson, New Engl. J. Med., 2002,
346, 476.
8
9
M. -Y. Jia, L. -Y. Niu, Y. Zhang, Q. -Z. Yang, C. -H. Tung, Y. -F.
Guan and L. Feng, ACS Appl. Mater. Interfaces., 2015, 7, 5907.
M. Marí, A. Morales, A. Colell, C. García-Ruiz and J. C.
Liu, X. -D. Zhou and X. -Q. Yu, Chem. Commun., 2015, 51
6781.
,
Fernandez-Checa, Antioxid. Redox Signaling., 2009, 11, 2685.
10 M. H. Lee, J. H. Han, J. -H. Lee, H. G. Choi, C. Kang and J. S.
Kim, J. Am. Chem. Soc., 2012, 134, 17314.
11 J. R. Casey, S. Grinstein, J. Orlowski, Nat. Rev. Mol. Cell Biol.
2010, 11, 50.
12 M. P. Murphy, Biochim. Biophys. Acta, 2008, 1777, 1028.
13 W. Zhang, W. Liu, P. Li, J. kang, J. Wang, H. Wang and B. Tang,
Chem. Commun., 2015, 51, 10150.
14 K. D. Belfield, M. V. Bondar, A. R. Morales, L. A. Padiha, O. V.
Przhonska and X. H. Wang, ChemPhysChem, 2011, 12, 2755
15 Z. G. Yang, N. Zhao, Y. M. Sun, F. Miao, Y. Liu, X. Liu, Y. H.
Zhang, W. T. Ai, G. F. Song, X. Y. Shen, X. Q. Yu, J. Z. Sun and
W. Y. Wong, Chem. Commun., 2012, 48, 3442.
16 M. H. Lee, N. Park, C. Yi, H. Han, J. H. Hong, K. P. Kim, D. H.
Kang, J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc.,
2014, 136, 14136.
17 J. -T. Hou, M. -Y. Wu, K. Li, J. Yang, K. -K. Yu, Y. -M. Xie and X.
-Q. Yu, Chem. Commun., 2014, 50, 8640.
18 Y. Liu, K. Li, M. -Y. Wu, Y. -H. Liu, Y. -M. Xie and X. -Q. Yu,
Chem. Commun., 2015, 51, 10236.
19 W. Xu, C. L. Teoh, J. Peng, D. Su, L. Yuan and Y. -T. Chang,
Biomaterials, 2015, 56, 1.
20 G. Masanta, C. S. Lim, H. J. Kim, J. H. Han, H. M. Kim and B. R.
Cho, J. Am. Chem. Soc., 2011, 133, 5698.
21 J. Yang, L. J. Giles, C. Ruppelt, R. R. Mendel, F. Bittner and M.
L. Kirk, J. Am. Chem. Soc., 2015, 137, 5276.
22 S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E. –H. Joe, B. R. Cho
and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915.
23 Y. Liu, F. Meng, Y. Tang, X. Yu and W. Lin, New J. Chem., 2016,
40, 3726.
24 C. S. Lim, G. Masanta, H. J. Kim, J. H. Han, H. M. Kim and B. R.
Cho, J. Am. Chem. Soc., 2011, 133, 11132.
25 W. Yang, P.S. Chan, M. S. Chan, K. F. Li, P. K. Lo, N. K. Mak, K.
W. Cheah and M. S. Wong, Chem. Commun., 2013, 49, 3428.
26 H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2013, 42, 6019.
27 X. Liu, Y. Sun, Y. Zhang, N. Zhao, H. Zhao, G. Wang, X. Yu, H.
Liu, J. Fluoresc., 2011, 21, 497.
28 W. Wang, O. Rusin, X. Xu, K. K. Kim, J. O. Escobedo, S. O.
Fakayode, A. Kristin, K. A. Fletcher, M. Lowry, C. M.
Schowalter, C. M. Lawrence, F. R. Fronczek, I. M. Warner and
R. M. Strongin, J. Am. Chem. Soc., 2005, 127, 15949.
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View Article Online
DOI: 10.1039/C6NJ00330C
In this work, we developed a dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging of
mitochondrial RSH in living system. In particular, the probe CA-TPP was capable of using TP fluorescence to track
mitochondrial RSH over a long period for the first time.