A dansyl-appended N-heterocycle for Cu2+ and S2? recognition via a displacement mode

Article abstract of DOI:10.1016/j.saa.2018.11.017

A novel L-proline based heterocycle 3 of C₂ symmetry has been designed and synthesized for cation and anion recognition in aqueous solution. Ligand 3 shows a strong affinity to Cu²⁺ ion, and their interaction induces a remarkable fluorescence quenching in DMSO:H₂O = 9:1 (HEPES buffer, 0.01 M, pH 7.4) among various metal ions. Both the in-situ generated and isolated 3-Cu²⁺ complex exhibit specific fluorescence recovery upon addition of S^2?, even in the presence of S₂O₃ ^2?, L-histidine, and thiol-containing amino acids. For this dual functional switch, a combination of ¹H NMR titration, ESI mass and FT-IR spectra suggest that its sensing behavior is via a displacement mode. Sequential “on-off-on” fluorescence bio-imaging of the heterocycle 3 to Cu²⁺ and S^2? was carried out in HeLa cells.

Full text of DOI:10.1016/j.saa.2018.11.017

Accepted Manuscript
A dansyl-appended N-heterocycle for Cu2+ and S2− recognition
via a displacement mode
Xu Wang, Peng Xia, Xiaohuan Huang
PII:
DOI:
Reference:
S1386-1425(18)31014-X
https://doi.org/10.1016/j.saa.2018.11.017
SAA 16581
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 19 July 2018
Revised date:
Accepted
date:
8 November 2018
12 November 2018
Please cite this article as: Xu Wang, Peng Xia, Xiaohuan Huang , A dansyl-appended
N-heterocycle for Cu2+ and S2− recognition via a displacement mode. Saa (2018),
https://doi.org/10.1016/j.saa.2018.11.017
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ACCEPTED MANUSCRIPT
A dansyl-appended N-heterocycle for Cu2+ and S2- recognition via
a displacement mode
Xu Wang, Peng Xia, Xiaohuan Huang
Hubei Key Laboratory of Pollutant Analysis and Reuse Technology, College of Chemistry and
Chemical Engineering, Hubei Normal University, Huangshi 435002, PR China
Abstract
A novel L-proline based heterocycle 3 of C2 symmetry has been designed and
synthesized for cation and anion recognition in aqueous solution. Ligand 3 shows a
strong affinity to Cu2+ ion, and their interaction induces a remarkable fluorescence
quenching in DMSO:H2O = 9:1 (HEPES buffer, 0.01 M, pH 7.4) among various metal
ions. Both the in-situ generated and isolated 3-Cu2+ complex exhibit specific
2-
fluorescence recovery upon addition of S2-, even in the presence of S2O3 , L-histidine,
and thiol-containing amino acids. For this dual functional switch, a combination of 1H
NMR titration, ESI mass and FT-IR spectra suggest that its sensing behavior is via a
displacement mode. Sequential “on-off-on” fluorescence bio-imaging of the
heterocycle 3 to Cu2+ and S2- was carried out in HeLa cells.
Keywords: amide-containing heterocycle; S2- recognition; displacement mode; cell
imaging
1. Introduction
The design and development of novel chemical sensors for cations, anions and
biomolecules recognition have been a hot topic in supramolecular chemistry during the
past decades [1–3]. Transition metal ions have received extensive attention due to their
essential role in living organisms [4,5]. Copper is the third most abundant transitional
metal in the human body after iron and zinc, which plays a critical role in a variety of
metalloenzymes as a catalytic cofactor [6]. In humans, the lack of Cu2+ may affect cell
metabolism and enzymatic activities, whereas high levels of Cu2+ can damage the
 To whom correspondence should be addressed. Tel. +86-07146572179
E-mail address: xhuang@hbnu.edu.cn (Xiaohuan Huang)

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kidneys and liver and lead to various neurological diseases [7]. Actually, different types
of Cu2+ probes, e.g. carbon dots [8,9], metal-organic frameworks [10], nanoparticles
[11], and synthesized chemosensors [12–14] have been explored for CuII recognition.
Among them, fluorescent chemosensors display great advantage in high sensitivity,
high selectivity and simplicity [15,16]. Although the paramagnetic nature of Cu2+ ion
always results in fluorescence quenching upon binding, the Cu-complex has been
widely used in anion recognition as secondary sensor by a “switch-on” fluorescence
response [17–19].
Sulfide is a conventional toxic pollutant among different anions, which is always
released as a byproduct from some industrial processes, such as sulfuric acid conversion,
dyes, mining and leather industries [20–23]. In addition, sulfide anion can also be
produced owing to the reduction of sulfates by microorganisms and the breakdown of
microorganisms containing sulfur proteins [24], which is more toxic once protonated
to form HS- and H2S. Continuous exposure to high concentrations of sulfide can lead
to various physiological problems such as suffocation, loss of consciousness and
respiratory paralysis [25]. Therefore, the rapid and sensitive detection of sulfide is of
great importance in environmental monitoring and treatments. A number of detection
techniques have been reported for sulfide recognition, such as electrochemical methods
[26,27], ion chromatography [28,29], inductively coupled plasma atomic emission
spectrometry (ICP-AES) [30], and fluorescence spectroscopy [31–35].
To our knowledge, a series of fluorescent sensors bearing cyclic binding moieties, such
as simple crown ethers, aza-crown ethers, and even azathia crown ether have been
developed for metal ion, anion, and bio-molecule recognition [36,37]. However, the
incorporation of amino acids to construct cyclic ligand has not been widely reported.
Actually, as major components of proteins, amino acids have exhibited versatile
abilities to form complexes with a variety of neutral molecules and ions. Thus, chiral
L-proline is introduced to construct an oxo polyamine macrocycle, which is expected
to bear steric hindrance and the dual features of azacrowns and oligopeptides [38,39].
We have reported a L-proline derived heterocycle appended with anthracene for cation
and histidine recognition [40]. In light of the fact that dansyl group usually shows a

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desirable property of an intense fluorescence in the visible region [3,41], a dansyl-
appended heterocyclic ligand was synthesized in this work. The obtained ligand shows
high affinity to Cu2+ in aqueous solution with fluorescence “switch off”. While 3-Cu2+
complex exhibits specific fluorescence “switch on” response to S2- with high sensitivity,
2-
even in the presence of S2O3 , histidine and thiol-containing amino acids. The cation
and anion recognition behavior of the rational designed receptor was applied in
biological imaging in living cells.
2 Experimental
2.1 Materials and instruments
All the reagents were bought from commercial suppliers and utilized without further
purification. The tetrabutylammonium salts of fluoride, chloride, bromide, iodide,
-
dihydrogenphosphate and the sodium salts of AMP, UMP, PPi, ClO-, NO2 and S2- were
purchased from Aldrich chemicals. HEPES, diethylenetriamine (DETA) and dansyl
chloride were used without further purifications. NMR spectra were measured on a
1
13
Varian Mercury 300 spectrometer operating at 300 MHz for H and 75 MHz for C
relative to tetramethylsilane as an internal standard.
2.2 Synthesis and structural characteristics
The synthetic procedures are listed in scheme 1. The structures of intermediates and the
desired product were identified by a combination of 1H NMR, 13C NMR and ESI-MS.
Scheme 1. Synthesis of ligand 3
Synthesis of compound 2. The L-proline derived chiral ester 1 of C2 symmetry was
synthesized according to the reported procedure [39]. Compound 1 (2.88 g, 0.01 mol)
was dissolved in anhydrous CH3OH (25 mL), and then added dropwise to a solution of

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diethylenetriamine (1.24 g, 0.012 mol) in anhydrous CH3OH (80 mL) in half an hour.
The mixture was stirred at 50°C for around 10 days under N2 protection, which was
monitored by thin-layer chromatography (TLC). The solvent was evaporated under
reduced pressure after the reaction completed. The crude product was purified by
recrystallization from anhydrous CH3CN to obtain compound 2 (0.45 g) in a yield of
14%. 1H NMR（CDCl3, 300MHz）σ ppm: 7.66 (s, NH, 2H), 3.59-3.73 (m, CH, 2H),
3.12-3.17 (m, CH2, 2H), 3.02-3.09 (m, CH2, 4H), 2.81-2.89 (m, CH2, 4H), 2.70-2.77
(m, CH2, 2H), 2.49-2.56 (m, CH2, 2H), 2.17-2.29 (m, CH2, 4H), 1.74-1.88 (m, CH2,
4H), 1.25-1.32 (m, CH2, 2H). MS (ESI): m/z 324.21 ([M+1]+, 100% ).
Synthesis of dansyl-appended heterocycle 3. A mixture of compound 2 (0.20 g, 0.62
mmol), dansyl chloride (0.17 g, 0.62 mmol) and triethylamine (0.12 g, 1.22 mmol) was
o
dissolved in 20 mL CH2Cl2 and stirred at 50 C overnight, which was monitored by
TLC. Until the reaction accomplished, the mixture was washed with water (3×20 mL).
The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to
dryness under reduced pressure to give a crude product, followed by purification using
silica-gel column chromatography (CH2Cl2/MeOH, 20:1, v/v) to obtain a yellow-green
solid (0.15 g, 43.49%). 1H NMR（CDCl3, 300 MHz）σ ppm: 8.56-8.53 (d, J = 9.00 Hz,
Ar-H, 1H), 8.43-8.40 (d, J = 9.00 Hz, Ar-H, 1H), 8.31-8.33 (d, J = 6.00 Hz, NH, 2H),
8.08-8.10 (d, J = 6.00 Hz, Ar-H, 1H), 7.49-7.59 (m, Ar-H, 2H), 7.18-7.20 (d, J = 6.00
Hz, Ar-H, 1H), 3.62-3.71 (m, CH, 2H), 3.30-3.78 (m, CH2, 8H), 3.21-3.23 (m, CH2,
2H), 2.98-3.01 (d, J = 9.00 Hz, CH2, 2H), 2.88(s, CH3, 6H), 2.66-2.69 (d, J = 9.00 Hz,
CH2, 2H), 2.35-2.44 (m, CH2, 2H), 2.10-2.14(m, CH2, 4H),1.78-1.85 (m, CH2, 4H). 13C
NMR (CDCl3, 75 MHz) ppm: 175.379, 151.799, 133.546, 130.597, 30.353, 130.199,
129.799, 128.418, 123.174, 119.353, 115.383, 68.391, 56.519, 54.508, 50.163, 45.371,
38.711, 29.782, 24.418. MS (ESI): m/z 557.21 ([M+1]+, 100%).
2.3 Cell cultures and imaging
HeLa cells were cultured in a medium consisted of 10% fetal bovine serum (FBS,
Sigma) and 80 μg/mL penicillin-streptomycin in a humidified 5% CO2 atmosphere at
37°C. Confocal fluorescence images were obtained by a TCS SP5 laser scanning

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microscope equipped with a 63× oil immersion objective (Leica, Germany).
3 Results and discussion
3.1. Sensing behavior of 3 towards metal ions
Firstly, the selective binding ability of ligand 3 towards metal ions was investigated
(Fig. 1a). When an equivalent amount of Cu2+ was added to 3 (DMSO:H2O = 9:1,
HEPES buffer, 0.01 M, pH 7.4), there was a remarkable fluorescence quenching. While
for other metal ions such as Al3+, Cd2+, Hg2+, Co2+, Zn2+, Ni2+, Pb2+, Eu3+, Te3+, Ca2+,
Ba2+ and Ag+ ions, no significant fluctuations of the fluorescence spectra were observed,
except a minor decrease upon Fe3+ addition. The fluorescence titration of 3 showed that,
the fluorescence intensity was reduced to 11.4% upon 1.0 equiv of Cu2+ addition (Fig.
1b). The photographs of ligand 3 after interaction with various metal ions (Fig. 1c)
suggest that 3 could be a selective fluorescent sensor for Cu2+ ion.
Fig. 1. (a) Fluorescence response of 3 upon addition of an equivalent amount of metal ions (Fe3+,
Al3+, Cd2+, Hg2+, Co2+, Zn2+, Ni2+, Pb2+, Ca2+, Ba2+, Ag+, Eu3+, Te3+ and Cu2+); (b) Fluorescence
titration profile of 3 upon addition of Cu2+ ions from 0 to 2.0 equiv. Inset: plot of fluorescence
intensity at 540 nm versus the equivalents of copper ions added. Measurement conditions: ligand 3
(50 μM) in DMSO:H2O = 9:1, HEPES buffer, 0.01 M, pH 7.4, ex = 328 nm, EX: 2; EM: 2. (c)
Photographs of 3 after addition of various metal ions (60 μM) taken under a UV lamp (λ = 365 nm).
The binding of ligand 3 to Cu2+ was further supported by ESI-high resolution mass

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spectrometry (HRMS) in the positive mode. Upon addition of Cu2+ into ligand 3, the
+
observed isotopic peak patterns of 3-Cu2 agree well with the calculated values as
shown in Figs. 2a and 2b. The results support the 1:1 stoichiometric complex formed
+
between ligand 3 and Cu2 . The association constant established by the Benesi-
Hildebrand equation is 1.32×104 M-1 [42], indicating that 3 has a strong affinity with
Cu2+. The detection limit of 1.39 μM was estimated by 3σ/k, where σ is the standard
deviation of the intercept of the regression line and the y-axis, and k is the slope of the
calibration curve between emission intensity versus Cu2+ concentrations [43]. It is less
than Cu2+ in freshwater guidelines in WHO (31.5 μM) [44].
Fig. 2. (a) Experimental isotope pattern of {(3 + Cu2+)-H}+ in ESI MS; (b) the calculated isotopic
peak pattern of the same complex.
3.2 Recognition behavior of 3-Cu2+ ensemble towards anions
Metal-heterocycle ensembles are utilized extensively for anion recognition due to the
fact that metal centre always serves as a strong coordination site for anion binding.
Firstly, the selectivity of 3-Cu2+ ensemble towards various anions, such as F-, Cl-, Br-,
-
-
-
I-, AcO-, H2PO4 , AMP, UMP, PPi, ClO4 , ClO-, NO2 , L-Cysteine (L-Cys), L-Histidine

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2-
(L-His), L-Cystine, S2O3 and S2- were investigated. As shown in Fig. 3a, a remarkable
fluorescence recovery was observed upon S2- addition. While the addition of other
anions, even the thiol-containing molecules like L-Cys and L-Cystine did not induce
2-
any fluorescence change, except a minor fluorescence enhancement induced by S2O3
(Fig. 3a). Further fluorescence titration demonstrated that the fluorescence of 3-Cu2+
ensemble recovered steadily upon the addition of S2- (Fig. 3b). It is worth noticing that
the isolated 3-Cu2+ complex exhibited comparable fluorescence titration results with
the in-situ generated 3-Cu2+ complex (see Supplementary information). Photographs
of 3-Cu2+ upon interaction with various anions (200 μM) clearly indicated an “off-on”
process with S2- (Fig. 3c). The detection limit was determined to be 6.1 μM according
to 3σ/k, less than the S2- in freshwater guidelines in WHO (14.8 μM) [28].
-
Fig. 3. (a) Fluorescence response of 3-Cu2+ upon addition of 4 equiv of S2-, F-, Cl-, Br-, I-, H2PO4 ,
-
-
2-
AMP, UMP, PPi, ClO4 , ClO-, NO2 , S2O3 , L-Cys, L-Cystine and L-His. (b) Fluorescence titration
profile of 3-Cu2+ upon addition of S2- from 0 to 6.4 equiv. (c) Photographs of 3-Cu2+ after addition
of various anions (200 μM) taken under a UV lamp (λ = 365 nm).
Competition experiments were conducted to further confirm the excellent selectivity
of 3-Cu2+. Although a similar heterocycle-Cu2+ complex appended with anthracene
has been reported for L-His recognition [40], the selectivity of 3-Cu2+ complex towards
S2- shows no interference by L-His and other potential interfering anions as presented
in Fig. 4. The reason is that sulfide ions are known to react with Cu2+ ions to form a

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low-solubility product CuS (Ksp = 6.3×10−36) [45], compared to Cu-L-His complex
with a stability constant of 1.26× 1010 [46].
Fig. 4. Fluorescence response of 3-Cu2+ in competitive experiments with different anions in
DMSO:H2O = 9:1 (HEPES buffer, 0.01M, pH 7.4). Black bars: 3-Cu2+ (50 μM). Red bars: 3-Cu2+
(50 μM) with other anions (200 μM). Blue bars: 3-Cu2+ (50 μM) with other anions (200 μM) and
S2- (200 μM).
The binding mechanism of heterocyclic ligand 3 with Cu2+ and S2- was verified by 1H
NMR titration experiments (Fig. 5). Upon addition of 0.5 and 1.0 equiv of Cu2+ into 3,
both the amide protons (N-H) at 8.11 ppm and aromatic hydrogen at 8.10 ppm shifted
downfield, and the fine structure of heterocycle 3 lost gradually because of the
paramagnetic property of Cu2+ ions. Notably, when 1.0 equiv of Cu2+ was added, the
integral area of N-H protons decreased to half, indicating a deprotonation process
occurred, consistent with the mass spectrum of 3-Cu2+ ensemble mentioned ahead.
Subsequently, when 0.5 and 1 equiv of S2- were added, the proton peaks of amide and
aromatic hydrogen shifted upfield, and a resumption of the fine structure of compound
3 was observed. The association constant of 3-Cu2+ complex is determined to be
1.32×104 M-1 as mentioned ahead. When S2- ion was added, it reacted with Cu2+
quickly and formed a stable CuS species [45], which released the ligand 3 from its
copper ensemble.

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Fig. 5. Partial 1H NMR (300 MHz) spectra of ligand 3 with titration of Cu2+ and S2- in DMSO-d6.
FT-IR spectra were recorded to further explain the “on-off-on” sensing mechanism of
3 towards Cu2+ and S2- (Fig. 6). The IR spectrum of 3 shows two characteristic bands
at 1663.97 and 1141.68 cm-1 assigned to ν(C=O) and ν(S=O), respectively. After
interaction with Cu2+, the two bands shifted lower frequencies at 1633.62 and 1096.92
cm-1, indicating that C=O and S=O groups were involved in Cu2+ coordination. While
after the addition of an equivalent amount of S2- to 3-Cu2+ complex, the stretching
vibrations of both C=O and S=O were shifted back to higher frequencies at 1660.86
cm-1 and 1140.12 cm-1, respectively, further confirming that the interaction between S2-
and 3-Cu2+ induces the release of ligand 3.

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Fig. 6. The FT-IR spectra data for 3, 3-Cu2+ complex and 3-Cu2+ complex upon S2- addition.
3.3 Fluorescence imaging in HeLa cells
To further investigate its application in biological system, the recognition behavior of
3 to Cu2+ and S2- recognition was carried out in HeLa cells. The cells were first stained
with ligand 3 (50 μM) in DMSO:DMEM = 1:99 at 37 °C for 30 min, followed by
washing with PBS buffer to remove the extracellular extra 3. Figs. 7A and 7a show the
confocal fluorescence and bright images of HeLa cells stained with 3, respectively,
clearly indicating that 3 is cell permeable. While after further exposure to Cu2+ (60 μM)
for 30 min, very weak intracellular fluorescence was observed (Fig. 7B). To show the
usage of its Cu2+ complex in sulfide recognition, the stained cells were further incubated
in S2- for 1 h, followed by 3 times PBS wash. Remarkable intracellular fluorescence
recovery was observed as illustrated in Fig. 7C. These results demonstrate that the
sequential “on-off-on” response of compound 3 to Cu2+ and S2- can be applied in cells.

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Fig. 7. Confocal fluorescence (A–C) in-situ images and bright field images (a–c) of Hela cells,
respectively. (A, a) were created after the addition of sole 3 (50 μM); (B, b) were created after the
addition of Cu2+ (60 μM); (C, c) were created after the addition of S2- (200 μM) to the in-situ
generated 3-Cu2+ complex.
4. Conclusions
A novel L-proline based heterocyclic ligand appended with dansyl group was designed
for the sequential recognition of Cu2+ and S2-. Firstly, ligand 3 shows strong affinity
towards Cu2+, probably because its size matches that of the artificial heterocycle.
Secondly, 3-Cu2+ complex exhibits specific recognition towards S2- with a detection
limit of 6.1 μM, even in the presence of other common interfering anions. For this dual
functional switch, its sensing behavior via a displacement mode was confirmed by a
1
combination of ESI mass, H NMR titration and IR spectra. The ligand 3 may have
potential applications for Cu2+ and S2- detection in environmental and biological
systems.

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Acknowledgments
We acknowledge the financial support from the National Natural Science Foundation
of China (NSFC) [Grant Nos. 21501055 and 11811520120] and Science and
Technology Foundation for Creative Research Group of HBDE (T201810).
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Graphical Abstract

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Highlights
1. A L-proline based heterocycle appended with dansyl was developed for Cu2+ and
S2- recognition.
2. Sequential “on-off-on” response to Cu2+ and S2- are successfully carried out in
HeLa cells.
3. A combination of 1H NMR titration, mass and IR spectra suggests that its sensing
behavior is via a displacement mode.