A near-infrared fluorescent probe based on a novel rectilinearly π-extended rhodamine derivative and its applications

Article abstract of DOI:10.1039/c9tb02739d

We designed and synthesized a novel near-infrared (NIR) mitochondria-Targeted fluorescent probe RQNA for the specific detection of mitochondrial Cu²⁺ because mitochondria are important reservoirs of intracellular copper. For the preparation of this probe, a novel π-extended fluorescent xanthene dye RQN was firstly synthesized via an intramolecular nucleophilic substitution of aromatic hydrogen (S_NAr^H) strategy. Then, probe RQNA was prepared by the reaction of RQN and hydrazine hydrate, followed by treatment with acetone. RQNA exhibited selectivity, sensitivity (22 nM), and fast response time (20 s) for the detection of Cu²⁺via a specific Cu²⁺-Triggered ring-opening and hydrolysis cascade reaction. RQNA is cell-membrane permeable and mitochondria-Targetable, and can be used for monitoring mitochondrial Cu²⁺ in living cells.

Full text of DOI:10.1039/c9tb02739d

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21 January 2018
Pages 341-528
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ISSN 2050-750X
PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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ARTICLE
A near-infrared fluorescent probe based on a novel rectilinearly π-
extended rhodamine derivative and its applications
Jin Gong, a Chang Liu, b Xiaojie Jiao, b Song He, b Liancheng Zhao a and Xianshun Zeng *,a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We designed and synthesized a novel near-infrared (NIR) mitochondria-targeted fluorescent probe RQNA for the specific
detection of mitochondrial Cu2+ because mitochondria are important reservoir of intracellular copper. For the preparation
of this probe, a novel π-extended fluorescent xanthene dye RQN was firstly synthesized via an intramolecular nucleophilic
substitution of aromatic hydrogen (SNArH) strategy. Then, the probe RQNA was prepared by the reaction of RQN and
hydrazine hydrate, and followed treatment by acetone. RQNA exhibited a selectivity, sensitivity (22 nM), and fast response
time (20 s) for the detection of Cu2+ via a specific Cu2+-triggered ring-opening and hydrolysis cascade reaction. RQNA is cell-
membrane permeable and mitochondria-targetable, and can be used for monitoring mitochondrial Cu2+ in the living cell.
were often utilized to design fluorescent probes for sensing
various biomolecules and metal cations due to their excellent
photophysical properties and biocompatibility, such as
Introduction
Copper is a redox-active metal that has a wide range of
applications in the chemical and electronics industries, and is
also an essential trace element playing important roles in
various physiological processes, such as cellular respiration,
maintenance of the immune system, bone health, etc.1 As a
cofactor for the respiratory enzyme cytochrome c oxidase,
mitochondria are important reservoir of intracellular copper.2
Therefore, humans must supplement their normal intake by
means of food. According to the World Health Organization
(WHO) and the Food and Agriculture Administration (FAA), the
average daily copper intake of adults should not exceed 10-12
mg.3 Studies have shown that abnormal levels of copper (Cu2+)
can cause various disease: When the level of Cu2+ in the body
is lower than normal, it will induce coronary heart disease,
arthritis, brain dysfunction, anemia and vitiligo;4 When the
level of Cu2+ is higher than normal, it will cause Parkinson's
disease, Alzheimer's disease, Wilson's disease, Menkes disease
and prion disease.5 Therefore, real-time monitoring Cu2+ levels
in mitochondria is important for early detection and
prevention of disease.
excellent water solubility, high quantum yields and tolerance
to photobleaching.7 However, most of the reported rhodamine
probes have short fluorescence emission wavelength (< 600
nm) and narrow Stokes shifts (< 30 nm), which limits their
intracellular imaging applications due to background
interference, self-quenching and photodamage to living cells.8
It has been reported that the use of near-infrared (NIR)
fluorescence imaging (650-900 nm) techniques can minimize
photodamage to living samples, increase tissue penetration
depth, and diminish signal interference caused by
autofluorescence of living samples.9 Meanwhile, fluorescent
dyes with large Stokes shifts can avoid self-quenching due to
back scattering from biological samples, resulting in precise
imaging and accurate sensing.10 Recently, Qian, Nagano, and
co-workers synthesized Si-rhodamines with emission
wavelengths in the NIR region, in which the bridging oxygen
atom in the xanthene moiety was replaced with a silicon
atom.11 However, these strategies still bear some limitations,
such as the narrow Stokes shifts.11 In fact, mitochondria-
targeted NIR fluorescent probes based on rhodamine scaffold
with larger Stokes shifts for specific detection of Cu2+ has not
been described. Therefore, designing and synthesizing
mitochondria-targeted NIR Cu2+ fluorescent probes based on
rhodamine structure with improved Stokes shifts remains a
huge challenge.
In recent years, the probe-based fluorescent imaging
technology has been introduced in the detection of Cu2+ due to
its high sensitivity, high selectivity, non-invasive and real-time
detection, and simplicity of implementation.6 Rhodamine dyes
a. School of Materials Science and Engineering, Harbin Institute of Technology,
Harbin, 150001, China.
Our group have revealed that rectilinearly extended π-
conjugate of rhodamine scaffold by engaging an unique
intramolecular nucleophilic substitution of aromatic hydrogen
(SNArH) strategy both effectively enlarged the emission
wavelength and Stokes shifts.12 Herein, a novel π-extended
xanthene dye RQN with NIR emissive wavelength and larger
Stokes shift was firstly prepared via the SNArH strategy by
b. Tianjin Key Laboratory for Photoelectric Materials and Devices, and Key
Laboratory of Display Materials and Photoelectric Devices, Ministry of Education,
School of Materials Science & Engineering, Tianjin University of Technology,
Tianjin, 300384, China. E-mail: xshzeng@tjut.edu.cn.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Fig. 1 Normalized absorption and fluorescence spectra of RQN and rhodamine B in
ethanol in the presence of TFA (1%).
Scheme 1 Synthesis of RQNA. Reagents and conditions: a) CH3I, CH3OH, K2CO3,
reflux, 22 h; b) 3'-(diethylamino)-7'-hydroxyspiro[isobenzofuran-1(3H),9'-
[9H]xanthen]-3-one, K2CO3, DMF, 80 °C, overnight; c) SnCl2, HCl, MeOH, reflux,
overnight; d) K2CO3, EtOAc, at room temperature, overnight; e) NH2NH2, 50 °C, 3
h; f) acetone, reflux, 6 h.
introducing the electron-accepting quinazoline moiety in
rhodamine scaffold to regulate the Stokes shift, and then the
probe RQNA was constructed by the mechanism of Cu2+-
induced ring-opening and hydrolysis cascade reaction. In
addition, the xanthene moiety is normally work as a functional
group for targeting mitochondria.13 The spirocyclic form of
RQNA is nonfluorescent, whereas the species of the Cu2+-
elicited ring-opening state gives rise to an obvious color
change and strong NIR fluorescence emission. Accordingly,
RQNA was an outstanding “turn-on” fluorescence probe with
NIR emission wavelength (656 nm), and could be used for
imaging of mitochondrial Cu2+ in living cells.
Scheme 2 Schematic illustration of the improved Stokes shift.
Optical properties of RQN
As depicted in Scheme S1, the dye was proposed to exist
in the forms of neutral (RQN) and protonated cation (RQN-H
and RQN-2H) in water under different pH conditions. To better
understand the structural conversions among them, the UV-
Vis and fluorescence spectra responses of RQN were firstly
investigated in water under different pH conditions. As shown
in Fig. S1a, RQN showed a maximum absorption band at 600
nm in acidic media (pH 1), indicating that it kept mainly in
RQN-2H. With the pH values increase (2-8), the absorption
band at and 606 nm decreased with the increase of pH value.
Meanwhile, a shoulder at ca. 576 nm appeared, which
suggested the inter-conversions between RQN-2H and RQN-H.
With the increase of pH values (8-12), a new absorption band
at ca. 698 nm was observed, indicated that it presented in the
form of RQN in basic condition. At the same time, the
enhanced fluorescence signal with increasing acidity may be
attributed to the enhanced ICT effect with the protonation of
the nitrogen atoms within the fluorophore (Fig. S1a, and
Scheme 2).
To better understand the photophysical properties of the
novel rectilinearly π-extended rhodamine derivative, the UV-
Vis absorption and fluorescence emission spectra of RQN were
measured in different solvents (CH3CN, DCM, DMF, DMSO,
EtOH, and H2O) in the presence of TFA (1%). As presented in
Table S1 and Fig. S2, the dye showed the maxima π-π*
transition absorption bands between 600 nm and 627 nm (ε ≥
10,900 M-1 cm-1) in all test solvents. Compared with the
maxima absorption band of rhodamine B at 553 nm in
ethanol,15 the rectilinearly π-extended RQN was red-shifted to
613 nm under the same condition. Upon excitation at 580 nm,
the maxima emission peaks of RQN range from 647 nm to 683
Results and discussion
Synthesis
The dye RQN and the probe RQNA were synthesized
according to the procedures in Scheme 1. As shown in Scheme
1, 7-chloro-6-nitroquinazolin-4(3H)-one (A) was converted to
its N-methylated derivative B in 89% yield by using MeI as
methylating reagent. By the reaction of 3'-(diethylamino)-7'-
hydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one14
with B, the compound C was obtained in 81% yield. Followed
by the reduction of the nitro group in the compound C by
using SnCl2 as reductive reagent in methanol, the ester
derivative of D was obtained in 77% yield. By using the unique
SNArH substitution reaction in the presence of base under mild
condition developed in our group,12 the intramolecular ring-
closed new dye RQN was yielded as a light yellow compound
(51%). With the reaction of RQN and hydrazine hydrate in
methanol, and followed treatment by acetone, the probe
RQNA was produced in 61% yield. The structures of the new
compounds B, C, D, RQN and RQNA were characterized by
using spectroscopic techniques, for example, 1H NMR, 13C NMR
and HRMS, and the spectra were provided in the supporting
information.
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nm were observed. Comparatively, the maxima fluorescence
emission band of RQN at 664 nm is significantly red-shifted
than that of rhodamine B at 572 nm in ethanol. On the other
hand, rhodamine B showed a narrow Stokes shift of 19 nm in
ethanol. However, the peak-to-peak Stokes shift (51 nm) of
RQN was obviously improved. It can be rationalzed that the
combination of the electron-withdrawing quinazoline group in
the π-extended xanthene fluorophore can regulate the
intramolecular charge transfer (ICT) processed. As shown in
Scheme 2, both rhodamine B and RQN bear a electron donor-
acceptor system from the electron-donating N,N-diethylamino
group to the electron-withdrawing 2'-methoxylcarbonylphenyl
group, which may produce a similar contribution in terms of
Stokes shifts. However, the donor-acceptor system in plane
from the electron-donating N,N-diethylamino group to the
electron-withdrawing quinazoline group in RQN can lead to
large changes in the energy difference between the ground
and the excited state, thereby producing an obviously
increased Stokes shifts.16 In addition, the fluorescence
quantum yields in these solvents were between 7.1% and
11.7%. Collectively, the rectilinearly π-extended RQN was a
novel NIR fluorophore with improved Stokes shift.
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DOI: 10.1039/C9TB02739D
Fig. 2 a) Fluorescence emission responses of RQNA (10 μM) upon addition of different
species (50 μM); b) Fluorescence ratio ((F-F0)/(FCu-F0)) changes of RQNA in the presence
of 50 μM Cu2+ and different species (50 μM). Metal cations: 1) Ag+; 2) Al3+; 3) Ca2+; 4)
Cd2+; 5) Co2+; 6) Cr3+; 7) Cu2+; 8) Fe2+; 9) Fe3+; 10) K+; 11) Li+; 12) Mg2+; 13) Mn2+; 14) Na+;
15) Ni2+; 16) Pb2+; 17) Pd2+; 18) Zn2+. The conditions: HEPES buffer (10 mM, pH = 7.4,
containing 20% CH3CN), λex = 575 nm, slit = 10/10 nm.
with Cu2+. As shown in Fig. S6, an intense peak at m/z
559.1989 corresponding to RQN-H was distinct after the
reaction of RQNA with Cu2+, suggesting that the Cu2+-elicited
ring-opening and hydrolysis cascade reaction of the probe is
correct.17 In order to confirm the stoichiometry between the
Cu2+ and RQNA, a Job’s plot was conducted (Fig. S7). The
analysis corresponding to 0.5, the concentration ratio
indicated the 1:1 stoichiometric ratio between RQNA and Cu2+.
Subsequently, a competitive experiment was conducted
to assess whether the background metal ions showed
interference or not with the detection of Cu2+. The responses
of RQNA toward Cu2+ in the presence of 5 equiv. coexistent
potentially interfering metal ions (Ag+, Al3+, Ca2+, Cd2+, Co2+,
Cr3+, Fe2+, Fe3+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Pd2+, and
Zn2+) were investigated under the same conditions. As shown
in Fig. 2b, the probe exhibited obvious response in the
presence of all of the tested foreign metal ions, revealing that
interfering metal species had minor or no interference to the
selective recognition properties of RQNA. Therefore, RQNA
has a high selectivity for Cu2+ in the presence of these tested
foreign metal ions.
Recognition properties of RQNA
In order to evaluate the recognition properties of RQNA,
selective experiments were performed. As shown in Fig. 2a,
the intensity of the fluorescence emission at 656 nm of the
probe was significantly enhanced after addition of 5 equiv. of
Cu2+. In contrast, no significant change in the fluorescence
emission spectrum was observed after addition of 5 equiv.
other metal ions, including Ag+, Al3+, Ca2+, Cd2+, Co2+, Cr3+, Fe2+,
Fe3+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Pd2+, and Zn2+.
Meanwhile, the similar enhancement trend as emission
spectrum was also exhibited in the absorption spectrum (Fig.
S3). Upon addition of Cu2+, the absorption intensity of the
probe at 598 nm was significantly enhanced. In contrast, no
obvious absorptive responses of the probe were observed
upon addition of other metal cations under the same
conditions. In addition, the spectral change process was also
accompanied by obvious color changes (Fig. S4). When all the
tested 18 metal ions were added separately into the solution
of RQNA, only the solution containing Cu2+ changed its color
from colorless to blue obviously which allowed the probe
could discriminate the Cu2+ ion from other metal cations. At
the same time, no significant change in the emission spectrum
was observed after addition of 5 equiv. reactive oxygen
species and reactive nitrogen species (Fig. S5). As shown in
Scheme S2, in the absence of Cu2+, RQNA mainly kept a non-
fluorescent spirocyclic form. The absorbance over 400 nm and
the fluorescence emission at 656 nm are very weak. In the
presence of Cu2+, however, the Cu2+-elicited ring-opening and
hydrolysis cascade reaction of the probe led to the intense
absorption at 598 nm and the fluorescence emission at 656 nm.
To verify the speculated mechanism, HRMS was conducted to
analyze the molecular weight of the probe after the reaction
Sensitivity of RQNA toward Cu2+
In order to further evaluate the sensitivity of the probe
towards Cu2+, we then performed continuous titration
experiments by using UV/Vis and fluorescence spectroscopy.
As shown in Fig. 3a, upon addition of increasing concentration
of Cu2+ (0-7 equiv.) to the solution, the fluorescent maximum
at 656 nm remarkably increased, which could be ascribed to
the Cu2+-induced ring-opening and hydrolysis cascade reaction
of the probe. Essentially, the fluorescence emission intensities
at 656 nm became constant when the amount of Cu2+ reached
5 equiv. Subsequent data analysis reveals an excellent linear
relationship (R = 0.9965) between the fluorescence intensity of
RQNA at 656 nm and the concentration of Cu2+ in the range of
0 to 25 μM (Fig. 3b). From the Cu2+ titration experiment, the
fluorescence dissociation constant (Kd) of RQNA for Cu2+ was
calculated to be 6.35 μM based on 1:1 stoichiometry (Fig. S8).
The detection limit of Cu2+ is determined to be 22 nM based on
the equation of 3σ/k, where k is the slope plotted from the
fluorescence intensity at 656 nm versus the concentration of
Cu2+, and σ is the relative standard deviation for 10 parallel
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Fig.
3 a) The fluorescence changes of RQNA (10 μM) treated with increasing
Fig. 4 The time course of the fluorescence intensities of RQNA (10 μM) at 656 nm after
treatment without or with Cu2+ (50 μM). The conditions: HEPES buffer (10 mM, pH 7.4,
containing 20% CH3CN), λex = 575 nm, slit = 10/10 nm.
concentrations of Cu2+ (0–70 μM). Inset: The plot of the fluorescence intensities at 656
nm versus the equivalents of Cu2+; b) The plot of the fluorescence intensities of RQNA
at 656 nm versus the concentrations of Cu2+ (0–25 μM). The conditions: HEPES buffer
(10 mM, pH 7.4, containing 20% CH3CN), λex = 575 nm, slit = 10/10 nm.
RQNA could selectively detect Cu2+ efficiently, the pH-
dependent experiments were performed. The pH-dependent
fluorescent responses of RQNA in the absence and presence of
Cu2+ were illustrated in Fig. S10. In the absence of Cu2+, RQNA
exhibited almost no obvious change in the fluorescence
intensity at 656 nm under a pH range of 4.0-12.0, indicating
that the probe is stable in a wide pH region. However, in the
presence of Cu2+, fluorescence intensity at 656 nm was
significantly enhanced over a pH range of 4.0-10.0 due to the
ring-opening induced by Cu2+, indicating that RQNA can be
utilized as a Cu2+ selective probe over a wide pH range. Above
results suggested that RQNA was qualified for the real-time
detection of trace amount of Cu2+ in the physiological
environment.
fluorescence measurements of the blank solution without Cu2+.
at 656 nm remarkably increased, which could be ascribed to
the Cu2+-induced ring-opening and hydrolysis cascade reaction
of the probe. Essentially, the fluorescence emission intensities
at 656 nm became constant when the amount of Cu2+ reached
5 equiv. Subsequent data analysis reveals an excellent linear
relationship (R = 0.9965) between the fluorescence intensity of
RQNA at 656 nm and the concentration of Cu2+ in the range of
0 to 25 μM (Fig. 3b). From the Cu2+ titration experiment, the
fluorescence dissociation constant (Kd) of RQNA for Cu2+ was
calculated to be 6.35 μM based on 1:1 stoichiometry (Fig. S8).
The detection limit of Cu2+ is determined to be 22 nM based on
the equation of 3σ/k, where k is the slope plotted from the
fluorescence intensity at 656 nm versus the concentration of
Cu2+, and σ is the relative standard deviation for 10 parallel
fluorescence measurements of the blank solution without Cu2+.
The detection limit was far lower than the maximum allowable
level of the World Health Organization (WHO) limit (30 μM)18
and the U.S. Environmental Protection Agency's (EPA) limit for
drinking water (20 μM)19. Meanwhile, the similar gradual
enhancement trend as emission spectrum was also exhibited
in the absorption spectrum (Fig. S9). Upon addition of
increasing concentration of Cu2+ (0-7 equiv.) to the solution,
the absorption intensity of RQNA at 598 nm remarkably
increased and reached the plateau when the accumulated
amount of Cu2+ reached 5 equiv. Upon addition of 5 equiv.
Cu2+, the quantum yield of RQNA was calculated to be 14.7%.
The results indicated that RQNA showed high sensitivity to
Cu2+ and could be used as a practical probe for quantitative
detection Cu2+ in drinking water.
Cell imaging
The spectral properties confirmed that RQNA had
advantages of high selectivity, high sensitivity and fast
response time for Cu2+ recognition, which encouraged us to
examine the potential applications of RQNA for imaging Cu2+ in
living cells. Before the imaging experiments in living cells, the
well-established MTT assay was adopted to study cytotoxicity
of the probe at varying concentrations (0, 0.1, 1, 5, 10, and 15
μM). The viabilities of HeLa cells were not noticeably affected
after cultured with 0-15 M RQNA for 24 h (Fig. S11),
indicating low cytotoxicity of RQNA. Then, bioimaging
applications of RQNA for monitoring of Cu2+ in living cells were
carried out (Fig. 5). HeLa cells were incubated with RQNA for
30 min in the growth medium, and then washed three times
with PBS to remove excess probes in the growth medium. As
expected, the HeLa cells stained with only RQNA showed
almost no fluorescence signals in the red channel (Fig. 5a),
confirming the extremely low background of the probe. In
contrast, after the addition of Cu2+ and then incubation for
another 30 min under the same conditions, the fluorescence
signals of the probe in the red channel increased obviously (Fig.
5b). Compared with the untreated cells, the relative
enhancement of fluorescence signals is about 75-fold (Fig. 5c).
The imaging results suggested that RQNA could readily
permeate the cell permeate and react with Cu2+, leading to the
formation of highly fluorescent product RQN-H in living cells.
By carefully observing the overlap images of the red
channel and the bright field, we found that the distribution of
the fluorescence signals in the cells unevenly fill the entire
cytoplasm, but concentrated in some regions, implied that
Time- and pH-dependent fluorescent responses of RQNA to Cu2+
In addition, time-dependent fluorescent responses of
RQNA to Cu2+ were evaluated at room temperature. As shown
in Fig. 4, after adding 5 equiv. of Cu2+, the fluorescence
intensity of RQNA at 656 nm rapidly increased and reached a
plateau within about 20 s, indicating that RQNA could be used
as an effective candidate for monitoring Cu2+ in real-time. In
contrast, in the absence of Cu2+, the fluorescence intensity of
RQNA at 656 nm remained substantially constant, revealing
that the probe had high photo-stability. At the same time, in
order to investigate the influence of the different pH on the
spectra of RQNA and the optimal working range in which
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florescence from other trackers (Lyso, ER, or Golgi) and Cu2+-
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DOI: 10.1039/C9TB02739D
activated RQNA was observed (Fig. 6b–d). Similarly, low
overlap coefficients and Pearson’s coefficients were found in
the intensity scatter plot, indicating that RQNA fitted not co-
localize exactly with these organelles. These results indicate
that RQNA can be used as an efficient organelle-targetable NIR
probe for mitochondrial Cu2+ imaging in living cells. Taken
together, considering its low cytotoxicity and the good
performance in cell imaging, the probe may be operated well
in monitoring the variation of Cu2+ concentration in the
mitochondria for the relevant diseases.
Fig. 5 Fluorescent images of HeLa cells with RQNA (1 μM). a) Treated with RQNA (1 μM)
for 0.5 h; b) RQNA pretreated HeLa cells with Cu2+ (5 μM) for 30 min; c) Histogram of
fluorescence enhancement. λex = 559 nm, λem = 618-718 nm.
Conclusions
In summary,
a
novel mitochondria-targeted NIR
fluorescent probe RQNA for the specific detection of
mitochondrial Cu2+ has been designed and synthesized. For
constructing this probe, a novel π-extended xanthene dye RQN
with NIR emissive wavelength and larger Stokes shift than
conventional rhodamine dye was firstly prepared via an
intramolecular nucleophilic substitution of aromatic hydrogen
(SNArH) strategy. RQNA was then prepared by the reaction of
RQN with hydrazine hydrate, and then with acetone. The
probe showed a selectivity, sensitivity, and fast response time
for the detection of Cu2+ via a specific Cu2+-elicited ring-
opening and hydrolysis cascade reaction. The probe is
mitochondria-targetable and can be used for monitoring
mitochondrial Cu2+ in the living cell.
Experimental
Instruments and reagents
All reagents and organic solvents used in the experiments
were commercially available in analytical grade and were used
directly without further purification unless otherwise stated.
The silica gel (200-400 mesh) used for chromatography were
purchased from Qingdao Ocean Chemicals. The structures of
the compounds were confirmed by NMR and high resolution
mass spectra (HRMS) spectrometry. NMR spectra were
obtained by Bruker spectrometer (400 MHz for 1H and 100
MHz for 13C) using tetramethylsilane (TMS) as an internal
standard. Chemical shift (δ) values are expressed in ppm and
coupling constants are expressed in Hz. HRMS were recorded
by Agilent 6510 Q-TOF LC/MS instrument (Agilent
Technologies, Palo Alto, CA). Spectroscopic properties were
collected on F-4600 fluorescence spectrophotometer (Hitachi
Japan) and UV-2550 UV/Vis spectrophotometer (Hitachi Japan).
The pH was measured using a FE 20/EL 20PH meter (Mettler-
Toledo Instruments (Shanghai) CO., Ltd.). Cell imaging was
carried out on an Olympus FV 1000-IX81 laser scanning
confocal imaging.
Fig. 6 Fluorescence imaging of the Cu2+-pretreated HeLa cells being costained with
RQNA (1 μM) and other organelle-targeting Trackers (200 nM). a) Costained with Mito-
Tracker Green; b) Costained with Lyso-Tracker Green; c) Costained with Golgi-Tracker
Green; d) Costained with ER-Tracker Green. Red channel: λex = 559 nm, λem = 618-718
nm. Green channel: λex = 488 nm, λem = 504-546 nm.
RQNA was likely to target organelles. According to the
literature, Cu2+ mainly accumulates in the mitochondria and
lysosome in living cells,20 and the xanthene dyes leads to the
proactive accumulation in mitochondria,13 we therefore
speculated that RQNA was likely to accumulate in one of the
two organelles. To determine the targeting-organelle ability of
the probe, the co-staining experiments through co-incubating
the HeLa cells with Mito-Tracker Green (Mito), Lyso-Tracker
Green (Lyso), ER-Tracker Green (ER), and Golgi-Tracker Green
(Golgi), respectively, were carried out. Surprisingly, a high
overlap coefficient (0.93) and Pearson’s coefficient (0.92) was
found in the intensity scatter plot of channel 1 and channel 2,
suggesting RQNA distributed predominantly in mitochondria.
In addition, the intensity scatter plot allowed us to highlight
corresponding yellow pixels on simulated images in
accordance with the overlay image (Fig. 6a), and the changes
in the intensity profiles in the linear region of interest across
the cell were synchronous in the two channels (Fig. S10). In
contrast, obvious difference of the location between the
Synthesis procedure
Synthesis of Compound B
CH3I (426mg, 3 mmol) was added to a mixed methanol (20 mL)
solution of 7-chloro-6-nitroquinazolin-4(3H)-one A (450 mg, 2
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mmol) and K2CO3 (828 mg, 6 mmol), and then heated to reflux Synthesis of RQN
for 22 h with stirring. The reaction was monitored by TLC until
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DOI: 10.1039/C9TB02739D
The compound D (575 mg, 1 mmol) and K2CO3 (138 mg, 1mmol)
were stirred in ethyl acetate (6 mL) at room temperature
overnight. Then, the mixture was extracted with ethyl acetate
(20 mL × 3) and the collected organic layers were dried over
anhydrous sodium sulfate. After removing solvents, the crude
product was purified by column chromatography to give
compound RQN as a light yellow solid (292 mg) in 51% yield.
1H NMR (400 MHz, CDCl3):  8.33 – 8.24 (m, 2H), 7.81 (t, J = 7.5
Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.54 (s, 1H), 7.29 (d, J = 7.2 Hz,
1H), 7.18 (s, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 1.8 Hz, 1H),
6.86 – 6.80 (m, 2H), 6.29 (s, 1H), 3.68 (s, 3H), 3.62 – 3.56 (m,
4H), 3.53 (s, 3H), 1.32 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz,
CDCl3):  165.29, 157.79, 157.48, 155.97, 155.01, 148.29,
146.64, 145.55, 142.73, 142.14, 133.60, 133.19, 131.44, 130.49,
130.42, 130.04, 129.68, 127.63, 116.83, 115.88, 114.61, 114.44,
113.05, 112.34, 111.93, 110.58, 100.29, 96.48, 52.65, 49.97,
49.75, 49.54, 46.01, 34.18. HRMS m/z = 573.2138 calcd. for
C34H29N4O5+ [M]+, found: 573.2139.
the disappearance of A, and extracted with dichloromethane
(50 mL × 3). The organic layers were collected and dried over
anhydrous sodium sulfate and concentrated. The crude
product was obtained by concentration under vacuum, and
then purified by column chromatography to afford the
1
compound B as a light yellow solid (425 mg) in 51% yield. H
NMR (400 MHz, (CD3)2CO) δ 8.78 (s, 1H), 8.39 (s, 1H), 8.02 (s,
1H), 3.61 (s, 3H). 13C NMR (100 MHz, (CD3)2CO) δ 189.58,
181.62, 180.55, 160.72, 159.83, 153.98, 150.45, 147.13, 63.41.
HRMS m/z = 240.0176 calcd. for C9H7N3O3Cl+ [M]+, found:
240.0203.
Synthesis of Compound C
The compound B (478 mg, 2 mmol) and 3'-(diethylamino)-7'-
hydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one
(688.6 mg, 2.2 mmol) were stirred in DMF (6 mL) at 80 °C
overnight. After cooling to room temperature, the mixture was
extracted with DCM (50 mL × 3) and the collected organic
layers were dried over anhydrous sodium sulfate. After Synthesis of RQNA
removing solvents, the crude product was purified by column
Under stirring at 50 °C, hydrazine hydrate (50 μL) was added to
chromatography to afford the compound C as a yellow solid
(478 mg) in 81% yield. 1H NMR (400 MHz, CDCl3):  8.76 (s, 1H),
8.02 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.58
(t, J = 7.4 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 7.6 Hz,
1H), 7.16 (dd, J = 9.0, 2.8 Hz, 1H), 6.92 (s, 1H), 6.61 (d, J = 2.7
Hz, 1H), 6.56 (d, J = 8.9 Hz, 1H), 6.46 (d, J = 2.0 Hz, 1H), 6.37
(dd, J = 8.9, 2.2 Hz, 1H), 3.54 (s, 3H), 3.36 (q, J = 6.9 Hz, 4H),
1.17 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, CDCl3):  169.28,
159.77, 155.66, 152.69, 152.61, 152.17, 149.99, 149.65, 148.63,
139.14, 135.15, 129.84, 128.73, 126.70, 125.50, 125.06, 123.98,
123.20, 121.47, 120.05, 119.21, 116.20, 114.34, 112.97, 108.77,
104.16, 97.56, 83.37, 44.51, 42.16, 34.18, 33.91, 12.51. HRMS
m/z = 591.1800 calcd. for C33H27N4O7+ [M]+, found: 591.1893.
the methanol solution of RQN (230 mg, 0.4 mmol). After 3
hours of reaction, the solution changed from blue to light
yellow. The solution was concentrated under vacuum, then
dissolved in acetone and heated to reflux for 6 h. After
removing solvents, the crude product was purified by column
chromatography to give the compound RQNA as a light yellow
1
solid (149 mg) in 61% yield. H NMR (400 MHz, CDCl3):  7.93
(dd, J = 5.2, 3.0 Hz, 1H), 7.75 (s, 1H), 7.52 – 7.42 (m, 2H), 7.08
(dd, J = 5.1, 2.9 Hz, 1H), 6.84 (s, 1H), 6.66 (s, 1H), 6.54 (d, J =
8.6 Hz, 1H), 6.22 (d, J = 7.5 Hz, 1H), 6.16 (s, 1H), 6.11 (s, 1H),
6.05 (s, 1H), 3.45 (s, 3H), 3.19 (s, 4H), 2.07 (s, 3H), 1.98 (s, 3H),
1.08 (t, J = 6.4 Hz, 6H). 13C NMR (100 MHz, CDCl3):  174.78,
160.85, 160.59, 152.94, 151.47, 149.10, 148.58, 144.67, 138.35,
132.55, 131.57, 131.29, 130.08, 128.54, 128.41, 123.59, 123.31,
118.04, 113.18, 112.02, 111.00, 109.18, 108.11, 106.89, 106.87,
104.68, 100.95, 97.58, 65.92, 44.23, 34.15, 32.69, 25.46, 22.05,
12.65, 12.62. HRMS m/z = 612.2600 calcd. for C36H32N6O4+ [M]+,
found: 612.2599.
Synthesis of Compound D
Concentrated hydrochloric acid (1 mL) was added to a mixed
methanol solution of the compound C (1180 mg, 2 mmol) and
stannous chloride (2250 mg, 10 mmol), and then heated to
reflux with stirring overnight. After cooling to room
temperature, the mixture was extracted with DCM (50 mL × 3) Cell culture and fluorescence imaging
and the collected organic layers were dried over anhydrous
HeLa cells for imaging were cultured in DMEM medium
sodium sulfate. After removing solvents, the crude product
was purified by column chromatography to give the compound
supplemented with 10% (v/v) fetal bovine serum and
penicillin/streptomycin (100 μg/mL) in an atmosphere of 5%
CO2 at 37 °C. The cells were cultured for 2 hours to adhere
them to the 12-well plate, and then subjected to fluorescence
imaging experiments. Cells were incubated with 1 μM RQNA
for 0.5 h, and then further incubated with Cu2+ (5 μM) for 30
minutes. For co-localization experiments, HeLa cells was
incubated with RQNA (1 μM) and 200 nM other organelle-
targeting Trackers (Mito-Tracker Green (Mito), Lyso-Tracker
Green (Lyso), ER-Tracker Green (ER), or Golgi-Tracker Green
(Golgi)) for 30 minutes, and then further incubated with Cu2+
(5 μM) for 30 minutes. Cells were washed with PBS three
1
D as a yellow solid (885 mg) in 77% yield. H NMR (400 MHz,
CDCl3):  8.25 (d, J = 7.8 Hz, 1H), 7.85 – 7.76 (m, 3H), 7.74 –
7.65 (m, 2H), 7.60 – 7.53 (m, 2H), 7.34 (d, J = 7.9 Hz, 1H), 7.30
(d, J = 8.1 Hz, 1H), 6.97 (s, 1H), 6.87 (d, J = 2.7 Hz, 1H), 6.76 (s,
1H), 4.05 – 3.94 (m, 2H), 3.88 – 3.78 (m, 2H), 3.68 (s, 3H), 3.51
(s, 3H), 1.43 (t, J = 7.1 Hz, 3H), 1.37 (t, J = 7.0 Hz, 3H). 13C NMR
(100 MHz, CDCl3):  165.20, 160.63, 159.31, 159.27, 158.93,
152.33, 150.14, 149.97, 144.50, 141.28, 137.97, 133.64, 132.99,
132.81, 131.65, 131.03, 130.27, 129.47, 128.58, 122.76, 120.84,
119.77, 119.22, 118.13, 112.98, 110.06, 96.58, 52.81, 48.19,
47.77, 34.07, 13.90, 12.50. HRMS m/z = 575.2322 calcd. for
C34H31N4O5+ [M]+, found: 575.2303.
6 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry B
Journal Name
ARTICLE
X. Jiao, C. Liu, Q. Wang, K. Huang, S. He, L. Zhao and X. Zeng,
times, then for fluorescence imaging by confocal fluorescence
microscope.
View Article Online
Anal. Chim. Acta, 2017, 969, 49; (i) Liu C., Wang Q., Jiao X.,
DOI: 10.1039/C9TB02739D
Yao H., He S., Zhao L. and Zeng X., Dyes and Pigments, 2019,
160, 989.
8
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Acknowledgements
This work was sponsored by the National Natural Science
Foundation of China (NSFC 21907075; 21272172), and the
Natural Science Foundation of Tianjin City (19JCZDJC32400;
18JCQNJC75900).
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Journal of Materials Chemistry B
Page 8 of 8
View Article Online
DOI: 10.1039/C9TB02739D
A near-infrared fluorescent probe based on a novel rectilinearly
π-extended rhodamine derivative and its applications
Jin Gong, Chang Liu, Xiaojie Jiao, Song He, Liancheng Zhao and Xianshun Zeng
A novel NIR fluorescent probe RQNA based on a π-extended rhodamine derivative RQN for the
specific detection of mitochondrial Cu2+ has been synthesized.