Selective Cadmium Fluorescence Probe Based on Bis-heterocyclic Molecule and its Imaging in Cells

Article abstract of DOI:10.1007/s10895-021-02748-7

Fluorescence probes that selectively image cadmium are useful for detecting and tracking the amount of Cd²⁺ in cells and tissues. In this study, we designed and synthesized a novel Cd²⁺ fluorescence probe based on the pyridine-pyrimidine structure, 4-(methylsulfanyl)-6-(pyridin-2-yl)pyrimidin-2-amine (3), as a low-molecular-weight fluorescence probe for Cd²⁺. Compound 3 could successfully discriminate between Cd²⁺ and Zn²⁺ and exhibited a highly selective turn-on response toward Cd²⁺ over biologically related metal ions. The dissociation constant (K_d) and the limit of detection (LOD) of 5.4 × 10^{? 6}?mol L^{? 1} and 4.4 × 10^{? 7}?mol L^{? 1}, respectively, were calculated using fluorescence titration experiments. Studies with closely related analogs showed that the bis-heterocyclic moiety of 3 acted as both a coordination site for Cd²⁺ and a fluorophore. Further, the methylsulfanyl group of compound 3 is essential for achieving selective and sensitive Cd²⁺ detection. Fluorescence microscopy studies using living cells revealed that the cell membrane permeability of compound 3 is sufficient to detect intracellular Cd²⁺. These results indicate that novel bis-heterocyclic molecule 3 has considerable potential as a fluorescence probe for Cd²⁺ in biological applications.

Full text of DOI:10.1007/s10895-021-02748-7

Journal of Fluorescence (2021) 31:1161–1167
https://doi.org/10.1007/s10895-021-02748-7
ORIGINAL ARTICLE
Selective Cadmium Fluorescence Probe Based on Bis-heterocyclic
Molecule and its Imaging in Cells
Masayori Hagimori1,2 & Yasushi Karimine2 & Naoko Mizuyama3 & Fumiko Hara1 & Takeshi Fujino4 & Hideo Saji5
&
Takahiro Mukai6
Received: 23 February 2021 /Accepted: 7 May 2021
/ Published online: 13 May 2021
#
The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021, corrected publication 2021
Abstract
Fluorescence probes that selectively image cadmium are useful for detecting and tracking the amount of Cd2+ in cells and tissues. In
this study, we designed and synthesized a novel Cd2+ fluorescence probe based on the pyridine-pyrimidine structure,
4-(methylsulfanyl)-6-(pyridin-2-yl)pyrimidin-2-amine (3), as a low-molecular-weight fluorescence probe for Cd2+. Compound 3
could successfully discriminate between Cd2+ and Zn2+ and exhibited a highly selective turn-on response toward Cd2+ over
biologically related metal ions. The dissociation constant (Kd) and the limit of detection (LOD) of 5.4 × 10− 6 mol L− 1 and 4.4 ×
10− 7 mol L− 1, respectively, were calculated using fluorescence titration experiments. Studies with closely related analogs showed
that the bis-heterocyclic moiety of 3 acted as both a coordination site for Cd2+ and a fluorophore. Further, the methylsulfanyl group
of compound 3 is essential for achieving selective and sensitive Cd2+ detection. Fluorescence microscopy studies using living cells
revealed that the cell membrane permeability of compound 3 is sufficient to detect intracellular Cd2+. These results indicate that
novel bis-heterocyclic molecule 3 has considerable potential as a fluorescence probe for Cd2+ in biological applications.
Keywords Cd2+ Fluorescent probe Bis-heterocyclic molecule Low-molecular-weight Cell membrane permeability
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Introduction
soil-to-plant transference [1, 2]. The uptake of cadmium by
food is unavoidable and causes serious health problems, espe-
cially in kidneys, lungs, liver, and the nervous system [1, 3–5].
In addition, long-term exposure to cadmium is considered a
causative factor in lung, prostate, breast, and kidney cancers
because of its long biological half-life in humans (17–30
years) [3, 6–8]. Therefore, reliable methods that can detect
cadmium in living cells and tissues are urgently required.
To investigate heavy metal ions including Cd2+ in living
systems, several analytical methods, such as atomic absorp-
tion spectrometry and inductively coupled plasma (ICP) mass
spectrometry, have been commonly used [9, 10]. These
methods are suitable for measuring the total metal ion content
in tissues, but unsuitable for real-time monitoring in specific
cells or tissues. Alternatively, organic fluorescence probes are
useful for real-time detection and tracking trace amounts of
metal ions in living systems because of their selectivity and
sensitivity. Further, their fluorescence properties can be tuned
for targeting metal ions by chemical modification, which is an
added advantage [11, 12]. There have been efforts to develop
Cd2+-selective fluorescence probes consisting of a metal co-
ordination site and fluorophore [13–18]; however, only a few
are available owing to their poor selectivity, solubility, or cell
Cadmium, a toxic heavy metal that occurs naturally in soils
and minerals, can accumulate in rice and vegetables through
* Masayori Hagimori
hagimori@mukogawa-u.ac.jp
1
Faculty of Pharmaceutical Sciences, Mukogawa Women’s
University, 11-68 Koshien Kyubancho, Nishinomiya 663-8179,
Japan
2
Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1
Sakamoto, Nagasaki 852- 8501, Japan
3
Division of Medical Innovation, Translational Research Center for
Medical Innovation, 1-5-4 Minatojima-minamimachi, Chuo-ku,
Kobe 650-0047, Japan
4
Graduate School of Science and Engineering, Saitama University,
255 Shimo-Okubo, Sakura- ku, 338-8570 Saitama, Japan
5
Graduate School of Pharmaceutical Sciences, Kyoto University, 46–
29 Yoshida-Shimoadachi- cho, Sakyo-ku, Kyoto 606-8501, Japan
6
Department of Biophysical Chemistry, Kobe Pharmaceutical
University, 4-19-1 Motoyamakita Machi, Higashinada-Ku,
Kobe 658-8558, Japan

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J Fluoresc (2021) 31:1161–1167
membrane permeability. More importantly, the inability to
distinguish between Zn2+ and Cd2+ is a critical issue and hin-
ders the development of Cd2+-selective fluorescence probes
[13–16]. Zn2+ and Cd2+ exhibit comparable physical and
chemical properties because they belong to the same group
in the periodic Table [12]; therefore, only a few probes that
successfully discriminate Zn2+ and Cd2+ have been reported
[17, 18]. Although chemical modification is an effective meth-
od to overcome this issue, it yields a high molecular weight
compound with a complex chemical structure. Consequently,
the cell membrane permeability of fluorescence probes
worsens.
Bis-heterocyclic compounds such as 2,2’-bipyridine are
known as chelating ligands that form charge complexes with
several types of metal ions and are used as core structures for
drug-like molecules and fluorescence probes [19–22].
Previously, bis-heteroaryl core structures, which acted as the
coordination site for Zn2+ and fluorophores, have been
exploited as low-molecular-weight fluorescence probes for
Zn2+ [23–25]. These probes based on the bis-heteroaryl core
had molecular weights of less than 500 g mol− 1, which
showed clear chelation enhanced fluorescence (CHEF) effect
on target metal ions and enabled good cell membrane perme-
ability. In addition, a simple tuning of the electron density of
the core structure by introducing electron-donating or
electron-withdrawing groups greatly affected the intermolec-
ular charge transfer (ICT) state and metal selectivity including
Cd2+.
Synthesis of 4-(methylsulfanyl)-6-(pyridin-2-yl)
pyrimidin-2-amine (3)
To a solution of 3,3-bis(methylsulfanyl)-1-(pyridin-2-yl)prop-
2-en-1-one (1) (225 mg, 1 mmol) [26] in pyridine (5 mL),
guanidine carbonate (2) (270 mg, 1.5 mmol) was added and
the mixture was refluxed for 5 h. The reaction mixture was
poured into 100 mL of ice water, and the precipitate was
collected by filtration, washed with water, and dried over-
night. The product was recrystallized from methanol to give
3 (64 g, 29 % yield) as pale-yellow crystals. Mp 142–143 °C.
1 H NMR (CDCl3, 300 MHz) δ 2.54 (s, 3 H), 5.14 (brs, 2 H),
7.32 (dd, J = 1.2, 7.5 Hz, 1 H), 7.55 (s, 1 H), 7.77 (dd, J = 1.8,
7.5 Hz, 1 H), 8.26 (d, J = 7.8 Hz, 1 H), 8.65 (d, J = 4.8, Hz,
13
1 H).
C NMR (CDCl3, 75 MHz) δ 12.9, 105.2, 121.9,
125.1, 137.1, 149.5, 154.5, 162.1, 162.5, 173.1. MS m/z:
219 [M + H+]. HRMS calcd for C10H11N4S [M + H+]:
219.0704. Found: 219.0695.
Synthesis of 4-(methylsulfanyl)-6-phenylpyrimidin-2-
amine (5)
Compound 5 was prepared from 3,3-bis(methylsulfanyl)-1-
phenylprop-2-en-1-one (4, 224 mg, 1.0 mmol) and guanidine
carbonate (2) (270 mg, 1.5 mmol) in a manner similar to that
described for the synthesis of 3. Recrystallization from meth-
anol gave 5 (68 mg, 31 % yield) as pale-yellow crystals (mp
97–98 °C). 1 H NMR (CDCl3, 400 MHz) δ 2.52 (s, 3 H), 6.65
(s, 2 H), 7.03 (s, 1 H), 7.46–7.50 (m, 3 H), 8.06 (d, J = 7.6 Hz,
2 H). MS m/z: 217 [M+]. HRMS calcd for C11H11N3S [M +]:
217.0674. Found: 217.0666.
Herein, we have designed and synthesized a novel low-
m o l e c u l a r - w e i g h t C d 2 + f l u o r e s c e n c e p r o b e
(4-(methylsulfanyl)-6-(pyridin-2-yl)pyrimidin-2-amine, 3;
MW 218 utilizing pyridine–pyrimidine as a bis-heteroaryl
core structure, which functions as both a Cd2+ coordination
site and a fluorophore. Compound 3 showed sensitive sensing
ability toward Cd2+ and could successfully discriminate Cd2+
from other ions. Furthermore, fluorescence imaging study in-
dicated compound 3 had good cell membrane permeability to
apply for a cellular system.
Synthesis of 4-(pyridin-2-yl)pyrimidin-2-amine (7) [27]
After dissolving sodium (500 mg, 21.8 mmol) in a solution of
guanidine carbonate (2) (1350 mg, 7.5 mmol) in dry ethanol
(10 mL), 3-(dimethylamino)-1-(2-pyridyl)-2-propen-1-one
(6) (880 mg, 5 mmol) was added, and the mixture was
refluxed for 24 h. The reaction mixture was concentrated un-
der reduced pressure, and the residue was recrystallized from
methanol to give 7 (770 mg, 44 % yield) as pale-yellow crys-
tals (mp 136–137 °C). 1 H NMR (CDCl3, 300 MHz) δ 5.43
(brs, 2 H), 7.33 (dd, J = 1.2, 7.5 Hz, 1 H), 7.60 (d, J = 5.1 Hz,
1 H), 7.76 (dd, J = 1.8, 6.0 Hz, 1 H), 8.28 (d, J = 7.8 Hz, 1 H),
8.41 (d, J = 5.1 Hz, 1 H), 8.67 (d, J = 6.0 Hz, 1 H). 13 C NMR
(CDCl3, 75 MHz) δ 108.2, 121.7, 125.3, 137.1, 149.6, 154.6,
159.5, 163.4, 164.3. MS m/z: 173 [M + H+]. HRMS calcd for
C9H9N4 [M + H+]: 173.0827. Found: 173.0822.
Experimental
Materials and Instruments
1
All chemicals were of the highest purity available. H and
13 C NMR spectra were recorded on Varian Gemini 300 and
JEOL ECP-400 NMR systems, and the chemical shifts are
reported in ppm. Mass spectra (MS) and HRMS were per-
formed using a JMS-700 spectrometer (JEOL, Tokyo,
Japan). UV-Vis absorption spectra were recorded using a
UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan).
Fluorescence spectra were collected using on a fluorescence
spectrophotometer (RF-5300PC, Shimadzu, Kyoto, Japan).
Spectroscopic Studies
A stock solution of each compound (1 × 10− 2 mol L− 1) was
prepared in DMSO. Solutions of metal ions were prepared by

J Fluoresc (2021) 31:1161–1167
1163
dissolving the perchlorate salts of metal ions (Na+, K+, Mg2+,
Ca2+, Fe2+, Fe3+, Ni2+, Zn2+, Cd2+, Co2+, Cu2+, Mn2+, and
Al3+) in deionized water. The fluorescence of each compound
(10− 6 mol L− 1) was analyzed in the absence and presence of
metal ions in EtOH/H2O (1:1, v/v). A Job’ plot was used to
investigate the binding stoichiometry of 3 to Cd2+. The disso-
ciation constant (Kd) was determined from a Benesi–
Hidebrand plot [28, 29].
The UV-Vis absorption spectra of 3 in EtOH/H2O
(1:1, v/v) were recorded during titration with Cd2+
(Fig. 1). The addition of Cd2+ altered the absorption
spectrum of 3 in a Cd2+-concentration-dependent man-
ner. Specifically, the absorption peaks at wavelengths
260 and 350 nm of 3 gradually increased with increas-
ing amounts of Cd2+. Figure 2 shows the course of the
fluorescence titration of 3 with Cd2+ in EtOH/H2O (1:1,
v/v). Free compound 3 showed fluorescence centered at
412 nm upon excitation at 334 nm. With the addition of
Cd2+, the fluorescence intensity gradually increased in a
Cd2+-concentration-dependent manner and the emission
maximum wavelength of 3 exhibited a bathochromic
shift of 8 nm, indicating that a CHEF effect occurred
with Cd2+. The fluorescence quantum yield of 3 was
2.2 × 10− 2 without Cd2+ and was increased to 3.9 ×
10− 2 by Cd2+ addition. Job’ plot analysis revealed that
the complex formed between compound 3 and Cd2+ had
a 1:1 stoichiometry (Fig. S1). The dissociation constant
(Kd) of this complex, as derived from the fluorescence
titration data using Benesi–Hildebrand Eqs. [28, 29],
was 5.4 × 10− 6 mol L− 1 (Fig. S2). In addition, the limit
of detection (LOD) of 3, calculated using LOD = 3σ/
slope, was 4.4 × 10− 7 mol L− 1. Thus, the sensitivity of
compound 3 is comparable to or better than those of
other reported Cd2+ fluorescence probes.
È
Â
à É
n
1=ðF−F0Þ ¼ 1= KaðFmax−F0Þ Cd2þ
þ 1=ðFmax−F0Þ
where F, F0, and Fmax are the fluorescence intensity, the fluo-
rescence intensity without Cd2+, and the fluorescence intensi-
ty in the presence of excess Cd2+, respectively. The associa-
tion constant (Ka) (the inverse of Kd) was determined from the
slope of the plot of 1/(F − F0) against 1/[Cd2+]. The selectivity
of 3 for Cd2+ was investigated using 10− 2 mol L− 1 metal
cation solutions. The effect of pH on the fluorescence proper-
ties of 3 was evaluated using various buffers:
tris(hydroxymethyl)aminomethane hydrochloride buffer (pH
3.0), acetate buffer (pH 4.0–5.0), and tris-hydrochloric acid
buffer (pH 6.0–8.0). The fluorescence quantum yield values
(Φ) were measured with respect to quinine sulfate solution
(Φ = 0.54) as standard.
The selectivity of 3 for Cd2+ detection was evaluated
by fluorescence spectrometry in the presence of various
cations (Al3+, Ca2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+,
Mn2+, Na+, Ni2+, and Zn2+) in EtOH/H2O (1:1, v/v). As
shown in Fig. 3, the fluorescence turn-on response was
only observed when Cd2+ was added to the solution of
3. Upon the addition of Zn2+, no fluorescence enhance-
ment was observed, indicating that compound 3 could
completely discriminate Cd2+ from Zn2+. Furthermore,
biologically relevant ions such as alkali ions (Na+ and
K+) and group 2 ions (Ca2+ and Mg2+) existing in the
millimolar range in living systems had insignificant ef-
fects on the fluorescence spectrum of 3. In the presence
of heavy metal ions (Co2+, Cu2+, Fe2+, Fe3+, Mn2+, and
Ni2+) and Al3+, the emission maximum peak showed a
bathochromic shift of approximately 10 nm and fluores-
cence quenching was observed. We also carried out
competition experiments between Cd2+ and other cations
(Fig. 4). After the addition of each metal ion (Al3+,
Ca2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+,
Cellular Imaging Using Fluorescence Microscope
Mouse macrophage-like cells (RAW264) were cultured in
Dulbecco’s modified Eagle’s medium that included 10 % fetal
bovine serum and 1 % penicillin at 37 °C in a humidified
atmosphere with 5 % CO2. The cells were incubated with 3
(100 µmol L− 1) in culture media for 30 min at 37 °C. After
washing with phosphate-buffered saline (PBS), the treated
cells were incubated with Cd(ClO4)2·6H2O (300 µmol L− 1
in culture media for 30 min at 37 °C. The incubated cells were
imaged using a fluorescence microscope BZ-X710 (Keyence,
Osaka, Japan).
)
Results and Discussion
Pyridine–pyrimidine compound 3 was synthesized with 29 %
yield via the one-pot reaction of 3,3-bis(methylsulfanyl)-
1-(pyridin-2-yl)prop-2-en-1-one (1) and guanidine carbonate
(2) in pyridine (Scheme 1).
Scheme 1 Synthesis of
compounds 3

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J Fluoresc (2021) 31:1161–1167
Fig. 1 Changes in the UV-Vis absorption of 3 (10− 5 mol L− 1) upon the
addition of Cd2+ (0–100 µmol L− 1) in EtOH/H2O (1:1, v/v)
Fig. 3 Fluorescence spectra of 3 (10− 6 µmol L− 1) and various metal
cations (100 µmol L− 1) in EtOH/H2O (1:1, v/v); excitation wavelength:
334 nm)
Ni2+, and Zn2+) to the solution of 3, the same amount
of Cd2+ was added. Fluorescence quenching was ob-
served in the presence of some heavy metal ions includ-
ing Ni2+, Cu2+, and Fe3+, indicating that these metal
ions interfere with the binding of Cd2+ to 3. In the
presence of other metal ions including alkali ions (Na+
benzene–pyrimidine analog 5 did not show a turn-on
response to Cd2+, likely due to the lack of a coordina-
tion site. Thus, the interaction of 3 with Cd2+ involves
the pyridine–pyrimidine moiety. Furthermore,
demethylsulfanyl analog 7 exhibited bathochromic shift
of 10 nm upon adding both Cd2+ and Zn2+, indicating
that the methylsulfanyl group at the 4-position of the
pyrimidine ring affects the ICT state and Cd2+ selectiv-
ity. Based on this result, we will develop related com-
pounds with different substituents at the 4-position of
the pyrimidine ring in the future.
and K+), group 2 ions (Ca2+ and Mg2+) and Zn2+
CHEF effects were observed with Cd2+. Although fluo-
rescence quenching was observed with Fe2+, Co2+
,
,
Mn2+, and Al3+, the fluorescence intensity increased
again upon adding Cd2+, indicating that these metals
were displaced by Cd2+. These spectroscopic data indi-
cated that compound 3 could be a selective probe for
Cd2+.
To evaluate the influence of pH on the Cd2+ detect-
ing ability of 3, we measured the intensity at the fluo-
rescent maximum wavelength (415 nm) in buffer solu-
tions at different pH ranges in the absence and presence
of Cd2+ (Fig. 6). The fluorescence of 3 at pH 3 de-
creased in the absence of Cd2+ by the protonation of
the nitrogen atom of free 3. However, the fluorescence
intensities of 3 in the absence and presence of Cd2+
The Cd2+ selectivity of compound 3 was investigated
using analogs 5 and 7, which are closely related to 3.
As shown in Scheme 2, benzene–pyrimidine analog 5
without a terminal basic nitrogen and demethylsulfanyl
analog 7 were prepared with 31 and 41 % yields, re-
spectively. Spectroscopic studies (Fig. 5) revealed that
Fig. 2 Changes in the fluorescence of 3 (10− 6 mol L− 1) upon the addition
of Cd2+ (0–100 µmol L− 1) in EtOH/H2O (1:1, v/v); excitation
wavelength: 334 nm
Fig. 4 Competitive binding experiments between various metal ions (10−
3 mol L− 1) and Cd2+ (10− 3 mol L− 1) with 3 (10− 6 mol L− 1) in EtOH/H2O
(1:1, v/v); excitation wavelength: 334 nm

J Fluoresc (2021) 31:1161–1167
1165
Scheme 2 Synthesis of the close
analogs (5 and 7)
Fig. 5 Fluorescence spectra of (a)
5 (excitation wavelength: 324
nm) and (b) 7 (excitation wave-
length: 346 nm) upon addition of
metal cations in EtOH/H2O (1:1,
v/v)
were nearly constant in the range of pH 4–8. These
results indicate that compound 3 can function as a
Cd2+ probe under physiological conditions. Thus, we
studied compound 3 via cellular imaging to evaluate
its cell membrane permeability.
Figure 7 shows the fluorescence microscopy images
of living cells (mouse macrophage-like cells; RAW264).
The cells cultured in the Dulbecco’s modified Eagle’s
medium was incubated with 3 (100 µmol L− 1) for
30 min at 37 °C in a humidified atmosphere of 5 %
CO2. After the cells were washed with PBS to remove
excess of 3, they exhibited a very weak fluorescence
signal (Fig. 7(a)). In contrast, the cells treated with 3
(100 µmol L− 1) for 30 min and Cd2+ (300 µmol L− 1
)
for another 30 min showed a bright intracellular fluo-
rescence signal (Fig. 7(b)). These data indicate that
compound 3 has good cell membrane permeability and
a fluorescence turn-on response to intracellular Cd2+. In
addition, no significant cytotoxicity was observed during
this study.
Conclusions
To develop a Cd2+ selective fluorescence probe with
cell membrane permeability, we synthesized low-
molecular-weight 3 (MW = 218) based on pyridine-
pyrimidine as a bis-heterocyclic core structure from the
one-pot synthesis of 3,3-bis(methylsulfanyl)-1-(pyridin-
2-yl)prop-2-en-1-one with guanidine carbonate in pyri-
dine and evaluated its fluorescence properties.
Compound 3 could successfully discriminate between
Fig. 6 Effect of pH on the fluorescence intensity of 3 in the absence (□)
and presence (■) of Cd2+

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J Fluoresc (2021) 31:1161–1167
Fig. 7 Fluorescence microscopy
images of RAW 264 cells. (a)
treated with 3 (100 µmol L− 1
)
only and (b) treated with both 3
(100 µmol L− 1) and Cd2+ (300
µmol L− 1
)
Cd2+ from Zn2+, and showed a Cd2+-selective fluores-
cence turn-on response in the presence of other biolog-
ically relevant metal ions. Moreover, fluorescence imag-
ing of living cells showed that the cell membrane per-
meability of compound 3 is sufficient to respond to
intracellular Cd2+. Thus, we expect that compound 3,
as a novel low-molecular-weight fluorescence probe for
Cd2+ will contribute to the selective detection and in-
vestigation of cadmium in biological and environmental
systems.
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Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s10895-021-02748-7.
Author Contributions All the authors (M. Hagimori, Y. Karimine, N.
Mizuyama, F. Hara, T. Fujino, H. Saji, T. Mukai) made substantial con-
tribution while preparing the manuscript.
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Funding This work was partly supported by the Konica Minolta Imaging
Science Encouragement Award of Konica Minolta Science and
Technology Foundation.
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Data Availability The data used to support the findings of this study are
included within the article.
Code Availability Not applicable.
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Conflict of Interest The authors declare that they have no conflict of
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