A novel near-infrared fluorescent probe with an improved Stokes shift for specific detection of Hg2+in mitochondria

Article abstract of DOI:10.1039/d0ob00507j

The mercury ion (Hg2+), one of the most notorious heavy metal ions, not only causes environmental pollution, but also endangers human health. There is evidence that Hg2+ tends to accumulate in the mitochondria and to induce apoptosis. However, mitochondria-targeted near-infrared (NIR) fluorescent probes with large Stokes shifts are still scarcely described for the specific detection of Hg2+. In this work, a novel near-infrared fluorescent probe JRQNS with a large Stokes shift (78 nm) was reported, and applied for sensitive and specific detection of Hg2+ in mitochondria by incorporating an additional amine group with fused rings to rhodamine dyes to enhance the electron donating ability of amine groups. As expected, the probe exhibited high selectivity and sensitivity to Hg2+ with a detection limit as low as 1.5 nM and fast response times (3 min), revealing that JRQNS could be used as a practical probe for quantitative detection of Hg2+ in real-time. Importantly, JRQNS can be used as an efficient organelle-targeting probe for imaging Hg2+ in the mitochondria of living cells, and thus detect Hg2+ in real-time there. The application of the probe for its selective localization in mitochondria along with the nanomolar level of limit of detection to Hg2+ ions provided a potential tool for studying the cytotoxic mechanisms of Hg2+. This journal is

Full text of DOI:10.1039/d0ob00507j

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Gong, C. Liu, X.
Jiao, S. He, L. Zhao and X. Zeng, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB00507J.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 7
View Article Online
DOI: 10.1039/D0OB00507J
ARTICLE
A novel near-infrared fluorescent probe with improved Stokes
shift for specific detection of Hg2+ in mitochondria
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
Mercury ion (Hg2+), one of the most notorious heavy metal ions, not only cause environmental pollution, but also endanger
human health. There is evidence that Hg2+ tend to accumulate in the mitochondria and to induce apoptosis. However,
mitochondria-targeted near-infrared (NIR) fluorescent probes with large Stokes shifts are still scarcely described for the
specific detection of Hg2+. In this work, a novel near-infrared fluorescent probe JRQNS with large Stokes shift (78 nm) was
reported, and applied for sensitive and specific detection of Hg2+ in mitochondria by incorporating an additional amine group
with fused rings to rhodamine dyes to enhance the electron donating ability of amine groups. As expected, the probe
exhibited high selectivity and sensitivity to Hg2+ with detection limit as low as 1.5 nM and fast response times (3 min),
revealing that JRQNS could be used as a practical probe for quantitative detection of Hg2+ in real-time. Importantly, JRQNS
can be used as an efficient organelle-targeting probe for imaging Hg2+ in mitochondria of living cells, and thus detect Hg2+ in
real-time there. The application of the probe for its selective localization in mitochondria along with the nanomolar level of
limit of detection to Hg2+ ions provided a potential tool for studying the cytotoxic mechanisms of Hg2+.
DOI: 10.1039/x0xx00000x
implementation, real-time and in situ detection, etc.16-30
Generally, derivatives of rhodamine were widely introduced in
Hg2+ detection due to excellent photophysical properties,
Introduction
As one of the most notorious heavy metal ions, mercury ion
(Hg2+) has attracted extensive attention and been considered as
the primary environmental hazard in the past few decades.1-3
Mercury species are a particular concern in many aspects due
to their extremely toxicity, which are subsequently bio-
accumulated through the food chain in the tissues of fish and
marine mammals and eventually enter the human body.4-6
Because mercury shows a strong affinity to sulfur-containing
organic ligands, it causes the dysfunction of proteins and
enzymes and results in a wide variety of diseases related to liver
and kidney dysfunction, central nervous system damage,
imbalance in the immune system, and even death.7-10
Furthermore, studies had shown that Hg2+ tend to accumulate
in the mitochondria, and induce apoptosis due to its binding to
thiol-containing proteins on the mitochondrial inner
membrane.11-15 Therefore, the development of mitochondria-
targeted detection tools with high selectivity and sensitivity is
important for studying the cytotoxic mechanism induced by
Hg2+.
including high quantum efficiency, high photostability, excellent
water solubility and biocompatibility.31-35 However, traditional
rhodamine dyes have some major drawbacks, such as the short
fluorescence emission wavelength (< 600 nm) and the narrow
Stokes shifts (< 30 nm), which limit their biological imaging
applications due to background interference, self-quenching,
excitation wavelength interference and photodamage to living
cells.31-35 To date, several strategies for increasing the emission
wavelength of rhodamine dyes have been developed. Generally,
extending the π-conjugated system of rhodamine scaffold is a
versatile strategy for shifting the excitation/emission bands of
fluorophores.36-37 In addition, replacing the oxygen atom in the
rhodamine scaffold with other atoms, including nitrogen, silicon,
selenium, tellurium, sulfur, antimony, etc, is another effective
strategy.38-39 However, these strategies still bear some
limitations, such as the narrow Stokes shifts, and few near-
infrared (NIR) Hg2+ fluorescent probes with mitochondrial
targeting.40-42 Consequently, it is necessary to design and
synthesize mitochondria-targeted fluorescent probes with NIR
emission and large Stokes shifts for monitoring Hg2+ in living
cells.
In recent years, small molecule fluorescent probe
technology had been widely used in the detection of Hg2+ due
to its high selectivity, high sensitivity, simplicity of
It is well known that the absorption and emission
wavelengths of traditional rhodamine dyes are highly
dependent on the substituents on the amino group. In a
pioneering works, the introduction of a piperazine group in the
scaffold of rhodamine could red shift the emission wavelengths
by altering the intramolecular charge transfer (ICT) properties
of the chomophore.43-44 In 2013, Zhang et al. fused 4-diethyl-
1,2,3,4-tetrahydroquinoxaline into rhodamine scaffold to afford
a. School of Materials Science and Engineering, Harbin Institute of Technology,
Harbin, 150001, China.
b. Tianjin Key Laboratory for Photoelectric Materials and Devices, 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
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 7
ARTICLE
Journal Name
new rhodamine dyes by enhancing the electron donating
ability,43 which not only red shifted the emission wavelength,
but also effectively increased the Stokes shift. In 2018, Lucka
and Liu et al. reported the incorporation of a piperazine fused
ring on the rhodamine scaffold, which also both increased the
emission wavelength and the Stokes shift.44 In addition, the
cationic xanthene moiety is normally work as a functional group
for targeting mitochondria,45 and increase the water solubility
of the probe. In this work, we reported a novel mitochondria-
targeted Hg2+ fluorescent probe JRQNS with NIR emission and
large Stokes shift by incorporating an additional amine group
with fused rings to rhodamine dyes to enhance the electron
donating ability of amine groups. As expected, the probe
exhibited high selectivity, high sensitivity, low detection limit
(1.5 nM), and fast response time (3 minutes) for the detection
of Hg2+ via a specific Hg2+-promoted desulfurization-cyclization
reaction. Gratifyingly, JRQNS can be accumulated in
mitochondria, and thus detect Hg2+ in real-time there, which
provided a potential tool for studying the cytotoxic mechanisms
of Hg2+.
View Article Online
DOI: 10.1039/D0OB00507J
Fig. 1 a) Fluorescence spectra of JRQNS (10 μM) in response to 1 equiv. various
metal ions. Inset: Color change of JRQNS in the absence/presence of Hg2+; b)
Fluorescence responses of JRQNS (10 μM) to Hg2+ (1 equiv.) in the presence of
other metal ions (1 equiv.). Metal ions: 1) Al3+; 2) Cr3+; 3) Fe3+; 4) Cd2+; 5) Co2+; 6)
Mg2+; 7) Cu2+ 8) Fe2+; 9) Ca2+; 10) Pd2+; 11) Zn2+; 12) Hg2+; 13) Pb2+; 14) Ni2+; 15)
Mn2+; 16) K+; 17) Ag+; 18) Li+; 19) Na+. The conditions: HEPES buffer (10 mM, pH =
;
7.4, containing 20% CH3CN), λex = 580 nm, slit = 5/5 nm.
JRQ, JRQN and JRQNS were fully characterized by NMR and
HRMS, and detailed data were recorded in the supporting
information.
Sensing properties of JRQNS
In order to test the in vitro response of JRQNS as a potential
Hg2+ sensor, absorption and emission spectra were recorded in
the presence of various metal ions under the same conditions,
including: Al3+, Cr3+, Fe3+, Cd2+, Co2+, Mg2+, Cu2+, Fe2+, Ca2+, Pd2+,
Zn2+, Pb2+, Ni2+, Mn2+, K+, Ag+, Li+, Na+, and Hg2+. As shown in Fig.
1a, the fluorescence intensity of the probe at 691 nm was
significantly enhanced after addition of 1 equiv. Hg2+, which is
significantly red shifted compared with those of conventional
rhodamine dyes (~570 nm),49 due to the electron donating
ability of amine groups was enlarged after introduction of the
Results and discussion
Synthesis of JRQNS
The fluorescent probe JRQNS was synthesized according to
the procedures in Scheme 1. The intermediate compounds 1, 2
and 3 were prepared according to the literature in high
yields,43,46-47 and the detailed reaction reagents and conditions
were depicted in Scheme 1. By using the rearrangement
reaction in the presence of methanesulfonic acid, the
intramolecular ring-opened new dye JRQ was yielded as a blue
compound (81%). With the reaction of JRQ and hydrazine
hydrate in methanol, the key intermediate JRQN was obtained
in 53% yield. Followed by the reaction of JRQN with phenyl
isothiocyanate under mild condition, the probe JRQNS was
produced in 55% yield. The structures of the new compounds
4-diethyl-1,2,3,4-tetrahydroquinoxaline.
In
contrast,
biologically relevant metal ions as well as other heavy-metal
ions did not afford any change in the emission spectra of JRQNS
upon metal ion addition, indicating that Hg2+ specifically
induced fluorescence recovery of the probe. Accordingly, when
1 equiv. Hg2+ ions were added, the absorption intensity of the
probe at 613 nm was significantly increased, whereas no
obvious changes of absorption intensity of the probe at 613 nm
were observed after addition of other metal ions (Fig. S1).
Simultaneously, upon addition of Hg2+, a colorimetric change
from colorless to dark blue was observed, which allowed the
probe could discriminate the Hg2+ ions from other metal cations
(inset of Fig. 1a). Gratifyingly, the Stokes shift of JRQNS was
found to be 78 nm, which was obviously larger than those of
conventional rhodamine dyes (< 30 nm).49
According to the literatures,48 we speculated that the
“turn-on” signal was initiated by Hg2+-promoted
desulfurization-cyclization reaction illustrated in Fig. S2. In the
absence of Hg2+, JRQNS mainly kept a non-fluorescent
spirocyclic form. The absorbance over 400 nm and the
fluorescence emission at 691 nm are very weak. In the presence
of Hg2+, however, the Hg2+-elicited desulfurization-cyclization
reaction of the probe led to the intense absorption at 613 nm
and the fluorescence emission at 691 nm. To verify the
speculated mechanism, HRMS was conducted to analyze the
molecular weight of the probe after the reaction with Hg2+. As
shown in Fig. S2, an intense peak at m/z 623.3126
corresponding to JRQNS-OX was distinct after the reaction of
Scheme 2 Synthesis route of JRQNS. Reagents and conditions: a) succinaldehyde,
CH3CN, 60 oC, overnight; b) NaBH4, toluene, AcOH, 5 oC to reflux, 5 h; c) toluene,
reflux, overnight; d) CH3SO3H, 80 oC, 8 h; e) NH2NH2, MeOH, 50 oC, 6 h; f) phenyl
isothiocyanate, DMF, 50 oC, 6 h.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
JRQNS with Hg2+, suggesting that the Hg2+-promoted
desulfurization-cyclization reaction of the probe is correct. To
further understand the sensing mechanism, the 1H NMR
titration experiments of JRQNS with Hg2+ were conducted in
DMSO-d6 (Fig. S3). Upon addition of Hg2+, two broad singlet of
thiourea group at δ 9.31 (Ha) and 8.71 (Hb) ppm completely
disappeared. Also, the aromatic protons Hc, He and Hf were
shifted upfield and Hd was shifted downfield after the addition
of Hg2+. These results basically supported our speculative
sensing mechanism. Furthermore, in order to confirm the
stoichiometry between the Hg2+ and JRQNS, a Job’s plot was
conducted (Fig. S4). The fraction of the maximum absorbance
of Hg2+ at 613 nm is 0.5, indicating that the binding formation
of stoichiometry is 1:1.
View Article Online
DOI: 10.1039/D0OB00507J
Fig. 2 a) Fluorescence spectra of JRQNS (10 μM) in the presence of different
concentrations of Hg2+ (0–1.1 equiv.); Inset: fluorescence intensity changes as a
function of Hg2+ concentration; b) The linear relationship between the emission
intensity and Hg2+ concentrations (0-2.5 μM); The conditions: HEPES buffer (10 mM,
pH = 7.4, containing 20% CH3CN), λex = 580 nm, slit = 5/5 nm.
Subsequently, in order to further evaluate the selectivity of
JRQNS towards Hg2+, the competition experiments were
performed in which Hg2+ ions were added to JRQNS upon
addition of 1 equiv. physiologically and environmentally
relevant potentially interfering metal ions under the same
condition. As shown in Fig. 1b, all of the tested interfering metal
ions had minor or no interference with signal response to Hg2+,
indicating that the presence of interfering ions did not affect the
selective identification of the probe. Therefore, the selectivity
and competition experiments results suggested that JRQNS
displayed excellent selectivity toward Hg2+ which afforded an
OFF-ON signal response, and could even expand the potential
applications in bioimaging related fields.
In order to further evaluate the sensitivity of the probe
towards Hg2+, thus, the continuous titration experiment was
conducted by using UV/Vis and fluorescence spectroscopy. As
shown in Fig. 2a, upon addition of increasing concentration of
Hg2+ (0-1.1 equiv.) to the solution, the fluorescence intensity of
the probe at 691 nm gradually increased and reached the
plateau at 1 equiv., which could be attributed to the Hg2+-
induced desulfurization-cyclization reaction. The binding
constant derived from the fluorescence titrations was found to
be 1.35 x 106 M-1 based on 1:1 stoichiometry.50 Subsequent data
analysis revealed an excellent linear relationship (R = 0.9990)
between the fluorescence intensity of JRQNS at 691 nm and the
concentration of Hg2+ in the range of 0 to 2.5 μM (Fig. 2b). The
detection limit of Hg2+ is determined to be 1.5 nM based on the
equation of 3σ/k,51-52 where k is the slope plotted from the
fluorescence intensity at 691 nm versus the concentration of
Hg2+, and σ is the relative standard deviation for 10 parallel
fluorescence measurements of the blank solution without Hg2+.
Compared with some of the other small molecule fluorescent
probes for detecting Hg2+, the detection limit of JRQNS was at a
relatively low level, and JRQNS exhibited lager Stokes shift and
faster response times (Table S1). The detection limit of JRQNS
was far lower than the U.S. EPA's (Environmental Protection
Agency) limit on drinking water (9.97 nM).53 Similarly, upon
addition of increasing concentration of Hg2+ (0-1.1 equiv.) to the
solution, the absorption intensity at 613 nm gradually increased
and reached the plateau when the accumulated amount of Hg2+
Fig. 3 a) Time-dependence fluorescence intensity of JRQNS (10 μM) upon addition
of 10 μM Hg2+; b) Fluorescence intensity of JRQNS (10 μM) as a function of pH
value in the absence/presence of Hg2+ (10 μM); The conditions: HEPES buffer (10
mM, pH = 7.4, containing 20% CH3CN), λex = 580 nm, slit = 5/5 nm.
Hg2+ and could be used as a practical probe for quantitative
detection Hg2+ in drinking water.
In addition, in order to assess the possibility of real-time
detection, we investigated the response time of the probe
towards Hg2+. In the absence of Hg2+, the fluorescence intensity
at 691 nm was maintained at the initial value and no obvious
change occurred within 10 minutes (Fig. 3a), indicating that
JRQNS could be stably present in the solution and owned high
photo-stability. In contrast, after adding 1 equiv. of Hg2+, the
fluorescence intensity at 691 nm increased rapidly and reached
a plateau within 3 minutes (Fig. 3a), indicating that JRQNS could
be used as an effective candidate for monitoring Hg2+ in real-
time. At the same time, for practical applicability in more
complicated systems, the response of the probe in the absence
and presence of Hg2+ at different pH values were evaluated. In
the absence of Hg2+, the probe exhibited weak fluorescence
under alkaline conditions due to the spirocyclic form, while high
fluorescence was observed under acidic conditions for ring-
opening induced by H+ (Fig. 3b). Upon addition of Hg2+, the
fluorescence intensity at 691 nm increased significantly over a
pH range of 5-10 (Fig. 3b) due to the ring-opening induced by
Hg2+, indicating that JRQNS can be utilized as a Hg2+ selective
probe over a wide pH range. Above results suggested that
JRQNS was qualified for the real-time detection of trace amount
of Hg2+ in the physiological environment.
Fluorescence imaging of JRQNS
Since JRQNS shows rapid response time, high photo-
stability, excellent selectivity and sensitivity, it is possible to
explore the potential applications of the probe for sensing Hg2+
in living cells. Before that, it is crucial to evaluate the
reached
1 equivalents (Fig. S5). Continuous titration
experiments revealed that JRQNS showed high sensitivity to
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 7
ARTICLE
Journal Name
enhancement of fluorescence signals is about 3-fold (Fig. 4c).
View Article Online
Thus, above-described results obviousDlyOI:r1e0v.1e0a3l9e/dD0tOhBa0t05t0h7eJ
probe had good membrane permeability and could be used for
imaging Hg2+ in living cells.
By carefully observing the overlay 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
JRQNS was likely to target organelles. As described above, Hg2+
mainly accumulates in the mitochondria,54-56 and the xanthene
dyes leads to the proactive accumulation in mitochondria,45
thereby JRQNS is highly likely to accumulate in mitochondria.
Furthermore, NIR fluorescent probes for detection of Hg2+ in
mitochondria are still rarely reported,40-42 which greatly
prompted us to further explore the localization properties of
the probe. To explore the targeting-organelle of JRQNS, JRQNS
was co-cultured with commercially available trackers Mito-
Tracker Green, Lyso-Tracker Green, ER-Tracker Green, and
Golgi-Tracker Green, respectively, and then cultured with Hg2+.
As shown in Fig. 5a, the fluorescence signals of JRQNS
completely overlapped with the fluorescence signals of Mito
and yellow pixels in the overlay image were observed, indicating
that JRQNS distributed predominantly in mitochondria. In
particular, high Pearson’s coefficient (0.93) and overlap
coefficient (0.93) indicated that the staining of JRQNS fitted well
with that of Mito. In contrast, the other overlay images showed
that the fluorescence signals of JRQNS and the fluorescence
signals of other trackers (Lyso, ER, or Golgi) were clearly
distributed in different regions (Fig. 5b-d). Similarly, the lower
Pearson’s coefficients and overlap coefficients showed that the
fluorescence signals of JRQNS and other trackers were
essentially not distributed in the same regions, indicating that
JRQNS fitted not co-localize exactly with other organelles.
These results indicated that JRQNS can be used as an efficient
organelle-targeting probe for imaging Hg2+ in mitochondria of
living cells, which provided a potential tool for studying the
cytotoxicity of Hg2+.
Fig.4 Fluorescence images of HeLa cells. a) Cells were incubated with JRQNS (0.1
μM) for 30 min; b) and then incubated with Hg2+ (0.1 μM) for another 30 min; c)
Histogram of fluorescence enhancement. λex = 559 nm, λem = 647-747 nm.
Fig.5 Fluorescence imaging of HeLa cells costained with JRQNS (100 nM) upon treatment
of and Mito-Tracker Green (200 nM), Lyso-Tracker Green Green (200 nM), ER-Tracker
Green (200 nM), or Golgi-Tracker Green (200 nM). Cells were incubated with probes at
37 °C for 30 min, and then treated with Hg2+ (100 nM) for another 30 min, and finally
washed before imaging. a) Costained with Mito-Tracker Green; b) Costained with Lyso-
Tracker Green, c) Costained with ER-Tracker Green; d) Costained with Golgi-Tracker
Conclusions
Green. Red channel: λex = 559 nm, λem = 664-764 nm. Green channel: λex = 488 nm, λem
511-534 nm.
=
In summary, we developed a novel near-infrared (691 nm)
fluorescent probe JRQNS with large Stokes shift (78 nm) by
integrating an additional amine group with fused rings to
rhodamine dyes to increase the electron donating ability of
amine groups. As expected, JRQNS exhibited high selectivity
and sensitivity to Hg2+ with detection limit as low as 1.5 nM and
fast response times (3 min), revealing that JRQNS could be used
as a practical probe for quantitative detection of Hg2+ in real-
time. Importantly, due to the cationic and lipophilic characters
of the xanthene moiety, the probe is mitochondria targetable.
JRQNS could permeate cell membranes and accumulate in the
mitochondria, and thus detect Hg2+ in real-time there, which
provided a potential tool for studying the cytotoxic mechanisms
of Hg2+.
cytotoxicity of JRQNS to living cells by a well-established MTT
assay. Fig. S6 depicted the viability of HeLa cells under various
probe concentrations from 0 μM to 20 μM. The results
demonstrate that more than 80% of cells are viable, showing
the low toxicity of the probe to cultured cells under
experimental conditions and inferring their potential use in
intracellular imaging of living cells. Then, bioimaging
applications of JRQNS for monitoring of Hg2+ in living cells were
carried out using HeLa cells. As shown in Fig. 4a, the probe
showed weak fluorescence signals under the red channel,
confirming the extremely low background of the probe. In
contrast, after the addition of Hg2+ and then incubation for
another 30 min under the same conditions, strong fluorescence
signals of the probe under the red channel (Fig. 4b) was
observed. Compared with the untreated cells, the relative
Experimental
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
Instruments and reagents
Hydrazine hydrate (50 μL) was added to the methVaienwoAlrsticoleluOtniloinne
(10 mL) of JRQ (1.02 g, 2 mmol), and then the reaction system
DOI: 10.1039/D0OB00507J
In this paper, all reagents and organic solvents were
commercially available in analytical grade, and were used
directly without further purification unless otherwise stated.
The structures of the compounds were confirmed by NMR and
was heated to 50 °C and reacted under vigorous stirring for 6
hours. After cooling to room temperature, the mixture was
extracted with DCM (50 mL × 3) and the collected organic layers
were dried over anhydrous Na2SO4. The crude product was
obtained by concentration under vacuum, and then purified by
column chromatography (DCM/MeOH, 8/1, v/v) to afford the
compound JRQN as a pale yellow solid (0.55 g) in 53% yield. 1H
NMR (400 MHz, CDCl3) δ 7.96 – 7.87 (m, 1H), 7.46 – 7.36 (m,
2H), 7.14 – 7.04 (m, 1H), 6.34 (s, 1H), 6.03 (s, 1H), 5.62 (s, 1H),
3.56 (d, J = 0.6 Hz, 2H), 3.40 – 3.29 (m, 4H), 3.13 (d, J = 7.6 Hz,
4H), 3.11 – 3.05 (m, 2H), 3.03 – 2.85 (m, 4H), 2.49 (qd, J = 15.9,
7.9 Hz, 2H), 2.08 – 1.98 (m, 2H), 1.86 (dt, J = 12.0, 6.2 Hz, 2H),
1.20 (t, J = 7.0 Hz, 3H), 0.83 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 166.25, 151.98, 149.03, 146.21, 143.53, 137.27,
132.32, 131.40, 129.79, 127.83, 123.94, 123.88, 122.87, 117.19,
107.70, 104.34, 104.24, 97.86, 66.90, 49.97, 49.51, 46.65, 45.74,
45.43, 45.35, 27.20, 22.02, 21.48, 21.28, 10.51, 9.61. HRMS m/z
= 522.2895 calcd for C32H35N5O2 [M+H]+, found: 522.2886.
1
HRMS spectrometry. H NMR (400 MHz) and 13C NMR (100
MHz) spectra were collected by Bruker spectrometer using
tetramethylsilane (TMS) as an internal standard. Chemical shift
(δ) values are expressed in ppm and coupling constants are
expressed in Hz. HRMS were measured by Agilent 6510 Q-TOF
LC/MS instrument (Agilent Technologies, Palo Alto, CA).
Spectroscopic properties were collected by F-4600 fluorescence
spectrophotometer (Hitachi Japan) and UV-2550 UV/Vis
spectrophotometer (Hitachi Japan). The pH was measured using
a FE 20/EL 20 pH meter (Mettler-Toledo Instruments (Shanghai)
CO., Ltd.). Cell imaging was performed by Olympus FV 1000-
IX81 laser scanning confocal imaging.
Preparation of stock solutions
Stock solutions (10 mM) of the nitrate salts of Al3+, Cr3+, Fe3+,
Cd2+, Co2+, Mg2+, Cu2+, Fe2+, Ca2+, Pd2+, Zn2+, Pb2+, Ni2+, Mn2+, K+,
Ag+, Li+, Na+, and Hg2+ ions were prepared in distilled water.
Stock solutions of JRQNS (5 mM) were prepared in DMF. Test
solutions were prepared by placing appropriate volume of the
stock solution into a 5 mL vial, adding an appropriate aliquot of
each metal stock, and diluting the solution to 3 mL with
CH3CN/HEPES (2/8, v/v). Unless otherwise stated, all data were
collected at 10 min after the addition of guest ions.
Synthesis of JRQNS:
Phenyl isothiocyanate (203 mg, 1.5 mmol) was added to the
DMF solution (10 mL) of JRQN (521mg, 1 mmol), and reacted
under an argon atmosphere at 50 °C for 6 hours. After cooling
to room temperature, the mixture was extracted with DCM (50
mL × 3) and the collected organic layers were dried over
anhydrous Na2SO4. The crude product was obtained by
concentration under vacuum, and then purified by column
chromatography (DCM/EtOAc, 20/1, v/v) to afford the
compound JRQNS as a pale yellow solid (361 mg) in 55% yield.
1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 7.4 Hz, 1H), 7.62 (d, J =
6.6 Hz, 2H), 7.59 – 7.53 (m, 1H), 7.20 (t, J = 7.6 Hz, 2H), 7.07 –
7.12 (m, 3H), 6.35 (s, 1H), 6.00 (s, 1H), 5.55 (s, 1H), 3.39 – 3.31
(m, 3H), 3.14 (dd, J = 11.8, 7.5 Hz, 3H), 3.08 (dd, J = 10.8, 6.0 Hz,
2H), 3.05 – 3.01 (m, 1H), 3.00 – 2.95 (m, 1H), 2.93 – 2.83 (m, 3H),
2.77 (dt, J = 14.2, 7.1 Hz, 1H), 2.50 – 2.34 (m, 2H), 2.05 – 1.98
(m, 2H), 1.80 (dt, J = 13.2, 7.0 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H),
1.21 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 167.32,
150.75, 149.46, 146.57, 144.14, 137.74, 134.07, 132.16, 128.99,
128.81, 128.24, 128.21, 125.07, 125.00, 124.96, 124.89, 124.67,
124.39, 117.63, 108.06, 106.00, 103.69, 130.64, 98.09, 49.83,
49.43, 46.65, 45.61, 45.39, 45.34, 27.32, 21.77, 21.25, 21.16,
14.21, 10.46, 9.74. HRMS m/z = 657.3011 calcd for C39H41N6O2S+
[M+H]+, found: 657.3017.
Synthesis
The key intermediate compounds 1, 2 and 3 were prepared
according to the literature.43,46-47
Synthesis of JRQ:
The compound 2 (2.2 g, 10 mmol) and 3 (3.7 g, 11 mmol) were
dissolved in 5 mL methanesulfonic acid, and then the reaction
system was heated to 80 °C and reacted under vigorous stirring
for 8 hours. After cooling to room temperature, the mixture was
extracted with DCM (50 mL × 3) and the collected organic layers
were dried over anhydrous Na2SO4. The crude product was
obtained by concentration under vacuum, and then purified by
column chromatography (DCM/MeOH, 15/1, v/v) to afford the
compound JRQ as a blue solid (4.1 g) in 81% yield. 1H NMR (400
MHz, CDCl3) δ 8.35 – 8.25 (m, 1H), 7.54 – 7.47 (m, 2H), 7.07 –
7.01 (m, 1H), 6.67 (s, 1H), 6.59 (s, 1H), 5.96 (s, 1H), 3.61 (s, 2H),
3.55 (dd, J = 14.2, 7.1 Hz, 2H), 3.42 – 3.37 (m, 4H), 3.19 (s, 2H),
3.07 – 2.91 (m, 4H), 2.64 – 2.51 (m, 2H), 2.02 (d, J = 5.0 Hz, 2H),
1.87 (d, J = 5.7 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.2 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 166.59, 155.76, 153.64,
151.59, 149.71, 146.43, 134.50, 134.42, 133.19, 131.65, 131.24,
129.41, 129.38, 125.79, 123.50, 114.85, 113.87, 104.64, 104.05,
94.41, 50.72, 50.26, 48.02, 47.02, 45.43, 44.04, 27.72, 20.72,
20.05, 19.89, 10.99, 9.42. HRMS m/z = 508.2600 calcd for
C32H34N3O3 [M]+, found: 508.2609.
Standard MTT assay
HeLa cells were inoculated with a 96-well plate and allowed to
adhere for 12 hours. After changing the culture medium,
different concentrations of JRQNS (0, 1, 3, 5, 10, and 20 μM)
were separately added to the cells and cultured for another 24
hours to allow them to fully enter the cells. The culture solution
was changed and washed three times with PBS, and then 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was
added to each well and cultured for another 4 hours. The
culture solution was removed and added DMSO, and then the
absorbance at 490 nm was measured after standing for 2 hours.
Synthesis of JRQN:
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 7
ARTICLE
Journal Name
20 D. Wu, A. C. Sedgwick, T. Gunnlaugsson, E. U. Akkaya, J. Yoon
Cell culture and fluorescence imaging
View Article Online
and T. D. James, Chem. Soc. Rev., 2017, 46, 7105.
DOI: 10.1039/D0OB00507J
HeLa cells for imaging were cultured in DMEM medium
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. The cells were incubated with 0.1 μM
JRQNS for 30 minutes, and then Hg2+ (0.1 μM) was added and
incubated for another 30 minutes. For co-localization
experiments, JRQNS (100 nM) were incubated with a 200 nM
trackers (Golgi-Tracker Green (Golgi), Mito-Tracker Green
(Mito), ER-Tracker Green (ER), and Lyso-Tracker Green (Lyso))
for 30 minutes, respectively, and then treated with Hg2+ (100
nM) for another 30 min. Fluorescence imaging was performed
after washing the medium three times with PBS buffer.
21 S. Wu, W. Tu, Y. Zhao, X. Wang, J. Song and X. Yang, Anal.
Chem., 2018, 90, 14423.
22 Q. Duan, H. Zhu, C. Liu, R. Yuan, Z. Fang, Z. Wang, P. Jia, Z. Li,
W. Sheng and B. Zhu, Analyst, 2019, 144, 1426.
23 L. Chen, S. J. Park, D. Wu, H. M. Kim and J. Yoon, Chem.
Commun., 2019, 55, 1766.
24 L. Li, Y. Wen, L. Xu, Q. Xu, S. Song, X. Zuo, J. Yan, W. Zhang and
G. Liu, Biosens. Bioelectron., 2016, 75, 433.
25 Y. Y. Liu, S. Naha, N. Thirumalaivasan, S. Velmathi and S. P. Wu,
Sens. Actuators. B Chem., 2018, 277, 673.
26 S. Li, D. Huang, J. Wan, S. Yan, J. Jiang and H. Xiao, Sens.
Actuators. B Chem., 2018, 275, 101.
27 Y. Zhang, H. Chen, D. Chen, D. Wu, Z. Chen, J. Zhang, X. Chen,
S. Liu and J. Yin, Sens. Actuators. B Chem., 2016, 224, 907.
28 Y. Shan, W. Yao, Z. Liang, L. Zhu, S. Yang, Z. Ruan, Dyes.
Pigment., 2018, 156, 1.
29 J. Tang, H. Wu, S. Yin and Y. Han, Tetrahedron. Lett., 2019, 60,
541.
30 B. Zhou, S. Qin, B. Chen and Y. Han, Tetrahedron. Lett., 2018,
59, 4359.
31 M. Beija, C. A. M. Afonso and J. M. G. Martinho, Chem. Soc.
Rev., 2009, 38, 2410.
Conflicts of interest
There are no conflicts to declare.
32 L. Yuan, W. Lin, K. Zheng and S. Zhu, Acc. Chem. Res., 2013, 46,
1462.
33 H. Zhu, J. Fan, B. Wang and X. Peng, Chem. Soc. Rev., 2015, 44,
4337.
34 G. Sivaraman, M. Iniya, T. Anand, N. G. Kotla, O. Sunnapu, S.
Singaravadivel, A. Gulyani and D. Chellappa, Coord. Chem.
Rev., 2018, 357, 50.
35 B. Kaur, N. Kaur, S. Kumar, Coord. Chem. Rev., 2018, 358, 13.
36 E. David, J. Lejeune, R. S. Pellet, J. Schulz, M. Lemaire and J.
Chauvin, Tetrahedron. Lett., 2008, 49, 1860.
Acknowledgements
This work was sponsored by the National Natural Science
Foundation of China (NNSFC 21907075; 21272172), and the
Natural Science Foundation of Tianjin City (19JCZDJC32400;
18JCQNJC75900).
37 C. Liu, X. Jiao, Q. Wang, K. Huang, S. He, L. Zhao and X. Zeng,
Chem. Commun., 2017, 53, 10727.
38 Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 5680.
Notes and references
1
M. F Wolfe, S. Schwarzbach and R. A. Sulaiman, Environ.
Toxicol. Chem., 1998, 17, 146.
39 W. Piao, K. Hanaoka, T. Fujisawa, S. Takeuchi, T. Komatsu, T.
Ueno, T. Terai, T. Tahara, T. Nagano and Y. Urano, J. Am. Chem.
Soc., 2017, 139, 13713.
40 H. Agarwalla, P. S. Mahajan, D. Sahu, N. Taye, B. Ganguly, S. B.
Mhaske, S. Chattopadhyay and A. Das, Inorg. Chem., 2016, 55,
12052.
41 J. Chen, W. Liu, B. Zhou, G. Niu, H. Zhang, J. Wu, Y. Wang, W.
Ju and P. Wang, J. Org. Chem., 2013, 78, 6121.
42 L. Lei, X. Tan, S. Luo and C. Shi, New. J. Chem., 2017, 41, 8899.
43 Z. Tian, B. Tian and J. Zhang, Dyes. Pigment., 2013, 99, 1132.
44 H. Liu, Y. Zhang, S. Xia, M. Fang, W. Mazi, Y. Zeng, T. Johnston,
A. Pap and R. L. Luck, Chem. Commun., 2018, 54, 7625.
45 Y. K. Kim, H. H. Ha, J. S. Lee, X. Bi, Y. H. Ahn, S. Hajar, J. J. Lee
and Y. T. Chang, J. Am. Chem. Soc., 2010, 132, 576.
46 E. A. Belyaeva, D. Dymkowska, M. R. Wieckowski and L.
Wojtczak, Toxicol. Appl. Pharmacol., 2008, 231, 34.
47 E. A. Belyaeva, T. V. Sokolova, L. V. Emelyanova and I. O.
Zakharova, Sci. World J., 2012, 2012, 1.
2
3
D. Zeller and S. Booth, Science, 2005, 310, 777.
F. D. Natale, A. Lancia, A. Molino, M. D. Natale, D. Karatza and
D. Musmarra, J. Hazard. Mater., 2006, 132, 220.
C. R. Baum, Curr. Opin. Pediatr., 1999, 11, 265.
M. Harada, Crit. Rev. Toxicol., 1995, 25, 1.
A. M. L. Kraepiel, K. Keller, H. B. Chin, E. G. Malcolm and F. M.
M. Morel, Environ. Sci. Technol., 2003, 37, 5551.
P. B. Tchounwou, W. K. Ayensu, N. Ninashvili and D. Sutton,
Environ. Toxicol., 2003, 18, 149.
4
5
6
7
8
9
D. W. Boening, Chemosphere, 2000, 40, 1335.
M. McNutt, Science, 2013, 341, 1442.
10 A. Renzoni, F. Zino and E. Franchi, Environ. Res., 1998, 77, 68.
11 N. Sone, M. K. Larsstuvold and Y. Kagawa, J. Biochem., 1977,
82, 859.
12 T. Toimela and H. Tahti, Arch. Toxicol., 2004, 78, 565.
13 A. C. Santos, S. A. Uyemura, N. A. G. Santos, F. E. Mingatto and
C. Curti, Mol. Cell Biochem., 1997, 177, 53.
14 P. B. Pal, S. Pal, J. Das and P. C. Sil, Amino. Acids., 2012; 42:
1669.
15 A. Tonazzi, N. Giangregorio, L. Console, M. Scalise, D. L. Russa,
C. Notaristefano, E. Brunelli and D. Barca, Indiveri C., Chem.
Res. Toxicol., 2015, 28, 1015.
48 S. K. Yao, Y. Qian, Z. Qi, C. Lu and Y. Cui, New J. Chem., 2017,
41, 13495.
49 M. Beija, C. A. M. Afonso and J. M. G. Chem. Soc. Rev., 2009,
38, 2410.
50 P. Laurent, H. Miyaji, S. R. Collinson, I. Prokeš, C. J. Moody, J.
H. R. Tucker and A. M. Z. Slawin, Org. Lett., 2002, 4, 4037.
51 M. Shortreed, R. Kopelman, M. Kuhn and B. Hoyland, Anal.
Chem., 1996, 68, 1414.
52 A. Caballero, R. Martinez, V. Lloveras, I. Ratera, J. Vidal-
Gancedo, K. Wurst, A. Tárraga, P. Molina and J. Veciana, J. Am.
Chem. Soc., 2005, 127, 15666.
16 E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108, 3443.
17 H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2012, 41, 3210.
18 Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2013, 113,
192.
19 Y. Tang, D. Lee, J. Wang, G. Li, J. Yu, W. Lin and J. Yoon, Chem.
Soc. Rev., 2015, 44, 5003.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
53 Mercury Update: Impact of Fish Advisories, EPA Fact Sheet
EPA-823-F-01-011; EPA, Office of Water: Washington, DC,
2001.
View Article Online
DOI: 10.1039/D0OB00507J
54 S. Kamino, M. Murakami, M. Tanioka, Y. Shirasaki, K.
Watanabe, J. Horigome, Y. Ooyama and S. Enomoto, Org. Lett.,
2014, 16, 258.
55 A. R. Jagtap, V. S. Satam, R. N. Rajule and V. R. Kanetkar, Dyes.
Pigment., 2009, 82, 84.
56 M. Konigsberg, N. E. López-Díazguerrero, L. Bucio and M. C.
Gutierrez-Ruiz, J. Appl. Toxicol., 2001, 21, 323.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins