Cd2+-triggered amide tautomerization produces a highly Cd2+-selective fluorescent sensor across a wide pH range

Article abstract of DOI:10.1016/j.dyepig.2016.06.017

An NBD-derived fluorescent sensor termed CdTS was reported to sense Cd²⁺ with very high binding selectivity and significant fluorescence turn-on signal selectivity (65 fold enhancement). The amide/di-2-picolylamine receptor binds Cd²⁺ in an imidic acid tautomeric form, but binds most of other metal ions in an amide tautomeric form. The transformable ability makes CdTS have the specific selectivity for Cd²⁺. Additionally, CdTS can fluoresently and colorimetricly recognize Cd²⁺ across a wide pH range from 4.5 to 11.5. Finally, we applied CdTS to detect Cd²⁺ in living cells.

Full text of DOI:10.1016/j.dyepig.2016.06.017

Accepted Manuscript
2+
2+
Cd –triggered amide tautomerization produces a highly Cd –selective fluorescent
sensor across a wide pH range
Yang Liu, Qinglong Qiao, Miao Zhao, Wenting Yin, Lu Miao, Liqiu Wang, Zhaochao
Xu
PII:
S0143-7208(16)30268-6
10.1016/j.dyepig.2016.06.017
DYPI 5301
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 21 April 2016
Revised Date: 8 June 2016
Accepted Date: 9 June 2016
2+
Please cite this article as: Liu Y, Qiao Q, Zhao M, Yin W, Miao L, Wang L, Xu Z, Cd –triggered amide
2+
tautomerization produces a highly Cd –selective fluorescent sensor across a wide pH range, Dyes and
Pigments (2016), doi: 10.1016/j.dyepig.2016.06.017.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
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CdTS can fluoresently recognize Cd across a wide pH range.

ACCEPTED MANUSCRIPT
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Cd –Triggered Amide Tautomerization Produces a Highly
2+
Cd –Selective Fluorescent Sensor across a Wide pH Range
a,b
b
b
b
b
a
Yang Liu, Qinglong Qiao, Miao Zhao, Wenting Yin, Lu Miao, Liqiu Wang*
b
and Zhaochao Xu*
aCollege of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao, Hebei 066004, China. E-mail:
liqiuwang@tom.com
bKey Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China. E-mail: zcxu@dicp.ac.cn
2
+
Abstract: An NBD-derived fluorescent sensor termed CdTS was reported to sense Cd with very
high binding selectivity and significant fluorescence turn-on signal selectivity (65 fold
2
+
enhancement). The amide/di-2-picolylamine receptor binds Cd in an imidic acid tautomeric form,
but binds most of other metal ions in an amide tautomeric form. The transformable ability makes
2
+
CdTS have the specific selectivity for Cd . Additionally, CdTS can fluoresently and
2+
colorimetricly recognize Cd across a wide pH range from 4.5 to 11.5. Finally, we applied CdTS
2
+
to detect Cd in living cells.
Keywords: Amide tautomerization; Fluorescent sensor; Cadmium ions; Wide pH range; Cells
imaging
1
Introduction
Cadmium is extremely toxic metals and can cause renal dysfunction, calcium metabolism
disorders and an increased incidence of cancers of the lung, prostate, pancreas, and kidney[1-3].
Its wide use in industry and agriculture lead to a high level of absorption and accumulation in
plants and other organisms, thus causing cadmium contamination. Although it has been
2
+
demonstrated that the uptake of Cd can affect cellular functions, the molecular mechanisms of
1

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2
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Cd -causing diseases remains unclear[4]. Fluorescent sensors are powerful tools to monitor in
vitro and/or in vivo biologically relevant species such as metal ions due to the simplicity and high
2
+
sensitivity of fluorescence[5-8]. Until now, a number of fluorescent sensors for Cd have been
2
+
reported with some successful applications to image Cd in living cells[9-33]. However, specific
2
+
2+
2+
binding selectivity for Cd over Zn and Hg in the same family as well as biologically
2+
3+
2+
abundant transition metal ions like Fe /Fe and Cu is still a challenge for fluorescent sensor
design. Cadmium speciation, adsorption and distribution in soils depends strongly on pH, with its
mobility decreasing with increasing alkalinity[34-35]. pH is also one of the most important
environmental factors determining cadmium bioavailability to organisms[34,36]. For example,
Bervoets et al. reported that cadmium uptake by the midge larvae Chironomus riparius increased
with increasing pH of exposure in the range of 5.5–9.0 but decreased between pH 9.0 and 10.0[37].
It has been widely reported that lowering environmental pH reduces cadmium toxicity in
bacteria[38-39]. For example, Worden et al. confirmed that cadmium was less toxic to Escherichia
coli at pH 5 than at pH 7 in M9 minimal salts medium[40]. Understanding mechanisms by which
pH mediates cadmium toxicity would be useful for minimizing cadmium toxicity in the
environment and for gaining insight into the interactions between organic and inorganic
components of life. Taking into account the complexity and uncertainty of environmental pH, a
fluorescent probe that is able to identify cadmium ions over a wide range of pH, covering acidic to
basic, will be very helpful to understand the bioavailability and toxicity of cadmium ions.
2
+
Unfortunately, these reported fluorescent sensors have the ability to identify Cd only in a narrow
pH range around neutral.
Most receptors for metal ions have a confined binding ‘cavity’. The high selectivity to an
2

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analyte is extremely difficult to achieve due to a single binding pattern. If the receptor is
transformable to bind the analyte of choice, that is the binding pattern of the receptor with analyte
is different from that with other competitors, the receptor may bind the analyst more specific and
favorable. In our previous work, we found an amide-containing DPA receptor shows extreme
2+
2+
selectivity for Zn attributing to their transformable ability[41-42]. The receptor binds Zn in an
imidic acid tautomeric form with highest affinity but most other HTM ions in an amide tautomeric
form. We conjugated this receptor to two different fluorophores naphthalimide and coumarin to
get zinc probes termed ZTRS[38] and CTS[42], respectively. In this paper, we introduced the
amide-DPA receptor to an NBD fluorophore. The newly synthesized compound CdTS displayed
2
+
2+
unexpected high binding selectivity for Cd rather than Zn along with a unique dramatic
1
fluorescence enhancement. The H-NMR and fluorescence studies reveal that the receptor binds
2
+
Cd in an imidic acid form. It is worth to notice that CdTS can fluoresently and colorimetricly
2
+
recognize Cd across a wide pH range from 4.5 to 11.5. CdTS was easily synthesized from
4
-Chloro-7-nitrobenzofurazan in three steps as shown in Scheme 1.
Scheme 1
2
Experimental
2
.1 Materials and instruments
Unless otherwise stated, all reagents were purchased from commercial suppliers and used without
1
13
further purification. H-NMR and C-NMR spectra were recorded on a VARIAN INOVA-500
spectrometer, using TMS as an internal standard. Mass spectrometry data were obtained with a
HP1100LC/MSD mass spectrometer and a LC/Q-TOF MS spectrometer. UV-visible spectra were
collected on a Perkin Elmer Lambda 35 UV/VIS spectrophotometer. Fluorescence measurements
3

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were performed on a VAEIAN CARY Eclipse fluorescence spectrophotometer (Serial No.
FL0812-M018).
2
2
.2 Synthesis
.2.1 Synthesis of 2.
To a solution of 1 (400 mg, 1 mmol) in MeOH (20 mL) was added ammonium hydroxide (4 mL,
8% in water) at room temperature. The reaction solution was stirred at room temperature for 24 h
2
under a nitrogen atmosphere. The solvent was evaporated in vacuo, then the crude product was
purified by silica gel chromatography with PE:EA = 1:1 to afford desired product as a brown solid
(
194 mg, 54% yield).
.2.2 Synthesis of 3.
A solution of 2-chloroacetyl chloride (146 mg, 1.30 mmol, 1.2 eq.) in 5 mL of dry CH Cl was
2
2
2
added dropwise to a solution of 2 (194 mg, 1.08 mmol) and 4-dimethylaminopyridine (DMAP)
171 mg, 1.40 mmol, 1.3 eq.) in 20 mL of dry CH Cl stirred in an ice bath. After stirred 2 h at
(
2
2
room temperature, the mixture was removed under reduced pressure to obtain a pale solid, which
was purified by silica gel column chromatography with PE:EA = 5:1 to afford desired product as a
1
yellow solid (119 mg, 43% yield). H NMR (500 MHz, CDCl ): δ 9.52 (s, 1H, NH), 8.58 (d, J =
3
1
3
8
1
2
2
3
.0 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H ), 4.35 (s, 1H, CH ); C NMR (125 MHz, CDCl ): δ 167.5,
2
3
+
45.9, 143.8, 136.1, 134.3, 130.8, 114.6, 43.9. HRMS (ESI): Calcd for C H ClN O [M+H]
8
6
4
4
57.0078; found 257.0080.
.2.3 Synthesis of CdTS.
(100 mg, 0.39 mmol), di-(2-picolyl)amine (DPA) (42 mg, 0.39 mmol), K CO (107 mg, 0.78
2
3
o
mmol), and potassium iodide (50 mg) were added to CH CN (50 mL). After stirring at 60 C for
3
4

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1
0 h under nitrogen atmosphere, the mixture was cooled to room temperature, and the mixture was
removed under reduced pressure ,then the residue was purified by silica gel column
chromatography (CH Cl :MeOH =100:1) to afford CdTS as a pale yellow solid (101 mg, 62 %
2
2
1
yield). H NMR (500 MHz, CDCl ): δ 12.37 (s, 1H, NH), 8.58 (d, J = 4.5 Hz, 2H), 8.53 (d, J = 8.0
3
Hz, 1H ), 8.45 (d, J = 8.0 Hz, 1H ), 7.62 (t, J = 7.5 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.17 (t, J =
1
3
6
.0 Hz, 2H), 4.05 (s, 4H), 3.62 (s, 2H) ; C NMR (125 MHz, CDCl ): δ 172.3, 157.6, 149.6,
3
1
45.4, 143.3, 136.8, 134.4, 134.3, 130.5, 123.2, 122.7, 112.8, 60.6, 58.9. HRMS (ESI): Calcd for
+
C H N O [M+H] 420.1420; found 420.1436.
2
0
18
7
4
2
.3 Culture of CHO cells and fluorescent imaging
CHO cells was hatched in an atmosphere of 5% CO and 95% air in Dulbecco’s modified
2
o
Eagle’s medium (DMEM, Invitrogen) at 37 C. The cells were seeded in 24-well flat-bottomed
o
plates and then incubated for 72 h at 37 C under 5% CO . 5 µM CdTS in the culture media
2
o
containing 0.1% (v/v) DMSO was added to the cells and the cells were incubated for 1 h at 37 C.
After washing twice to remove the remaining sensor, the cells were treated with 10 ꢀM Cd(ClO4)2
for 30 min. Fluorescence imaging was observed under a confocal microscopy (Olympus FV1000)
with a 60×objective lens.
3
Results and discussion
2
+
3
.1 Cd selectivity
The selectivity of the fluorescent response of CdTS to metal ions was first examined. CdTS
has a good water solubility, and in HEPES buffer at pH 7.4 (0.5% DMSO) displays very weak
2
+
emission centered at 555 nm (Φ = 0.005) upon excitation at 462 nm. Addition of 1 equiv of Cd
induces a bathochromic shift of the dominant emission band to 567 nm with a significant
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2
+
2+
2+
fluorescence increase (65-fold, Φ = 0.32) (Fig. 1a). The CdTS /Zn , CdTS /Hg and CdTS /Pb
complex showed slight enhanced emissions (Fig. 1a inset). The addition of other metal ions, such
+
+
+
2+
2+
2+
2+
2+
2+
3+
3+
+
2+
as Li , Na , K , Mg , Ca , Co , Ni , Cu , Fe , Fe , Cr , Ag and Pd , produced a negligible
change in the fluorescence spectra of CdTS (Fig. 1a). Thus, CdTS has a very high fluorescence
2+
selectivity for Cd .
Fig. 1
2
+
2+
It’s worth noting, even the changes were very small, that the binding of Zn and Hg
2
+
blue-shifted the emission to 548 nm and 552 nm, respectively, and the binding of Pb , similar to
2
+
Cd , red-shifted the emission to 563 nm (Fig. 1a inset). Inspired by the transformable sensing
mechanism of ZTRS and CTS which show blue-shifted emission in an amide tautomeric binding
form, while red-shifted emission in an imidic acid tautomeric form, we propose that CdTS binds
2
+
2+
2+
2+
Cd and Pb in an imidic acid tautomeric form, but Zn and Hg in an amide tautomeric
binding form. Subsequently, we performed competition experiments in the presence of 30 equiv of
+
+
+
2+
2+
2+
2+
2+
2+
2+
3+
3+
+
2+
Li , Na , K , Mg , or Ca , and 3 equiv of Co , Ni , Cu , Zn , Fe , Fe , Cr , Ag , Hg ,
2
+
2+
2+
Pb or Pd , with the subsequent addition of 1 equiv of Cd . As shown in Fig. 1b, the emission
2+
profile of the CdTS /Cd complex is unperturbed in the presence of alkali and alkaline earth
2
+
2+
cations. Of transition metal ions we tested, only Zn and Cu limit the turn-on response of CdTS,
2
+
indicating the strongest affinity and selectivity for Cd (Fig. 1b) over these metal ions. We believe
2
+
the specificity for Cd and unique fluorescence responses result from the transformable ability of
2
+
CdTS that is the displacement of other metal ions by Cd induces transformation of chelation
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2
+
from an amide to an imidic acid tautomeric form. Accordingly, the addition of Cd induced a
much more significant red-shift in absorption than other metal ions (Fig. 1c). Further studies
2
+
indicated the detection limit of CdTS for Cd is down to 10 nM.
3
.2 Binding mechanism
2
+
1
To confirm the imidic acid tautomeric binding form with Cd , we conducted H-NMR
titration experiments in DMSO-d6. The chemical shift of the amide NH can be used to distinguish
2
+
whether Cd is bound to carbonyl oxygen or imidic acid nitrogen. The complexation of the
carbonyl oxygen with metal ions blocks the amide resonance and then shifts the NH resonance
upfield. Correspondingly, the binding of the amide nitrogen with metal ions acts as an
electron-withdrawing group to shift the OH resonance downfield. As shown in Fig. 2a, the
resonance of the H4-9 protons undergo down-field shifts in DMSO with the addition of 1 equiv of
2
+
2+
Cd , which demonstrate the coordination of Cd with two pyridyl nitrogens and one aliphatic
2
+
amine nitrogen. With the addition of Cd , the chemical shift of the amide NH changed, which
2
+
indicated the coordination of Cd with the amide group. As aforementioned, the clear down-field
2
+
of H3 from 11.98 to 12.13 suggested that CdTS binds Cd in an imidic acid tautomeric form in
DMSO.
Fig. 2
2
+
Fig. 3 showed the fluorescence and absorption titration experiments of CdTS with Cd in
2
+
HEPES. When Cd was added to the solution of CdTS, a red-shifted emission with a maximum
at 567 nm was increased subsequently (Fig. 3a). The inset job plots in Fig. 3a indicated the
2
+
2+
CdTS/Cd complex had 1:1 stoichiometry. On addition of 1 equiv of Cd to the solution of
7

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CdTS, the absorbance at 400 nm decreased sharply to its limiting value, while the one at 462 nm
increases prominently with an isosbestic point at 424 nm, which induces a color change from
colourless to yellow (Figure 3b).
Fig. 3
2
+
3
.3 Effect of pH on Cd detection
2
+
The influence of pH on the detection properties of CdTS for Cd was then examined by
fluorescence titration in HEPES solution (Figure 4). From pH 4 to 12, the fluorescence intensities
2
+
of CdTS were all increased by the addition of Cd . Particularly between pH 4.5 and 11.5, the
fluorescence increase responses were significant, indicating the excellent fluorescent sensing
2
+
properties of CdTS for Cd in this pH range (Fig 4a). More importantly, the obvious colour
2
+
change to yellow and yellow fluorescence of CdTS in the prescence of Cd from pH 4.5 to 11.5
2
+
facilitate the Cd detection and expand the detection scope (Fig. 4b). Particularly, the probe CdTS
2
+
has a great potential to investigate the pH-dependent distribution and toxicity of Cd . CdTS is
also anticipated to help the understanding of mechanisms by which pH mediates cadmium
toxicity.
Fig. 4
2
+
3
.4 Cell imaging of Cd
We then sought to examine the Cd sensing properties of CdTS in living cells. CHO cells
treated with 5 µM CdTS alone exhibited very weak background fluorescence (Fig. 5a). The cells
incubated with 10 M Cd(ClO ) and CdTS displayed enhanced fluorescence (Fig. 5b). These
2
+
µ
4
2
2
+
experiments indicate CdTS can recognize intracellular Cd fluorescently. The cytotoxicity of
8

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CdTS was examined toward CHO cells by a MTT assay (Fig S7). The results showed that ˃ 90%
CHO cells survived after 24 h (5.0 ꢀM CdTS incubation), demonstrating that CdTS was of low
toxicity toward cultured cell lines.
Fig. 5
3
.5 Conclusion
In summary, we have developed an NBD-based fluorescent sensor CdTS for Cd recognition
2
+
2
+
which contains a transformable amide-DPA receptor. CdTS has the strongest affinity with Cd
2
+
among competitive metal ions and displays an excellent fluorescent selectivity for Cd with an
2
+
enhanced emission resulting from the Cd –triggered amide tautomerization. More importantly,
2
+
CdTS can fluoresently and colorimetricly recognize Cd across a wide pH range from 4.5 to 11.5,
2
+
which makes CdTS a candidate to investigate the pH-dependent distribution and toxicity of Cd .
2
+
However, Cd –triggered amide tautomerization in CdTS is beyond our expectation. In
2
+
conjunction with Zn –triggered amide tautomerization in ZTRS and CTS, we propose that
various metal ions may trigger the amide tautomerization of amide-DPA receptor in different
systems.
We thank financial supports from the National Natural Science Foundation of China
(21276251, 21506206, 21402191, 21502189), the 100 talents program funded by Chinese
Academy of Sciences, Dalian Cultivation Fund for Distinguished Young Scholars (2014J11JH130
and 2015J12JH205) and the National Science Fund for Excellent Young Scholars (21422606).
Supplementary data
9

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Supplementary data related to this article can be found at doi:10.1016/j.dyepig.XXXX.XX.XXX .
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41] Xu Z, Baek K-H, Kim HN, Cui J, Qian X, Spring DR, et al. Zn2+-Triggered Amide Tautomerization Produces a
Highly Zn2+-Selective, Cell-Permeable, and Ratiometric Fluorescent Sensor. J Am Chem Soc. 2010;132(2):601-10.
42] Xu Z, Liu X, Pan J, Spring DR. Coumarin-derived transformable fluorescent sensor for Zn2+. Chem Commun.
012;48(39):4764-6.
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Scheme 1
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Scheme 1 Synthesis of the fluorescent sensor CdTS.
Fig. 1 (a) Fluorescence spectra of 10 µM CdTS in the presence of various metal ions in aqueous
solution. (b) Fluorescence responses of CdTS to various metal ions in aqueous solution. Bars
represent the final fluorescence intensity at 567 nm over the original emission. White bars
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represent the addition of 3 equiv of metal ions (for Na , K , Mg and Ca , 30 equiv) to a 10 µM
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solution of CdTS. Black bars represent the subsequent addition of 1 equiv of Cd to the solution.
(c) Absorption spectra of 10 µM CdTS in the presence of various metal ions in aqueous solution.
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Fig. 2 H-NMR spectra of CdTS in the presence of Cd in DMSO-d6.
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Fig. 3 The fluorescence (a) and absorption (b) titration experiments of CdTS with Cd in HEPES.
The inset shows the Job plot evaluated from the fluorescence with a total concentration of 10 µM.
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Fig. 4 (a) Influence of pH on the fluorescence sensing of CdTS for Cd . (b) Colour changes and
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visible emission observed from samples of CdTS/Cd in different pH solutions.
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Fig.5 Fluorescence images of CHO cells incubated with 5 µM CdTS and Cd . Cells treated with
CdTS a) in the absence and b) presence of 10 µM of Cd(ClO ) . (c) bright field image.
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Cd can trigger amide tautomerization in the amide-DPA receptor.
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The probe is extremely sensitive to Cd with 65-fold emission enhancement.
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The probe has an excellent selectivity for Cd .
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The probe can recognize Cd across a wide pH range from 4.5 to 11.5.
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The probe can image Cd in living cells.