Single chemosensor for sensing multiple analytes: Selective fluorogenic detection of Cu2+ and Br?

Article abstract of DOI:10.1016/j.tetlet.2018.09.024

A tripodal receptor R1 with a combination of nitrogen and oxygen-based binding sites was designed and used for the selective determination of Cu²⁺. The fluorescence emission profile of R1 in the presence of Cu²⁺ showed a marked enhancement in fluorescence intensity, indicating high selectivity among other metal ions. The R1–Cu²⁺ complex was further explored as a sensor for anion detection. Upon addition of Br^?, switching to a fluorescence “off” state was observed. The Br^? selectivity of the complex over a wide range of concentrations was observed via titrimetric analysis through changes in the emission spectra.

Full text of DOI:10.1016/j.tetlet.2018.09.024

Accepted Manuscript
Single chemosensor for sensing multiple analytes: selective fluorogenic detec-
tion of Cu2+ and Br –
Yoon Gun Ko, Mayank, Narinder Singh, Doo Ok Jang
PII:
S0040-4039(18)31104-3
https://doi.org/10.1016/j.tetlet.2018.09.024
TETL 50263
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2018
4 September 2018
9 September 2018
Please cite this article as: Ko, Y.G., Mayank, Singh, N., Jang, D.O., Single chemosensor for sensing multiple
analytes: selective fluorogenic detection of Cu2+ and Br– , Tetrahedron Letters (2018), doi: https://doi.org/10.1016/
j.tetlet.2018.09.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Single chemosensor for sensing multiple analytes: selective
fluorogenic detection of Cu2+ and Br–
Yoon Gun Ko,a Mayank,b Narinder Singhb,* and Doo Ok Janga,*
aDepartment of Chemistry, Yonsei University, Wonju 26493, Republic of Korea;
bDepartment of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab 140001, India
*Corresponding authors. E-mail: nsingh@iitrpr.ac.in (N. Singh), dojang@yonsei.ac.kr (D. O. Jang).
Abstract
A tripodal receptor R1 with a combination of nitrogen and oxygen-based binding sites was designed and used
for the selective determination of Cu2+. The fluorescence emission profile of R1 in the presence of Cu2+ showed
a marked enhancement in fluorescence intensity, indicating high selectivity among other metal ions. The R1–
Cu2+ complex was further explored as a sensor for anion detection. Upon addition of Br–, switching to a
fluorescence “off” state was observed. The Br– selectivity of the complex over a wide range of concentrations
was observed via titrimetric analysis through changes in the emission spectra.
Keywords: tripodal, copper, bromide, fluorescence, chemosensor, receptor
1

1.
Introduction
The quantitative and qualitative analysis of elements in the environment is important to human health [1].
Copper is an essential element required in trace amounts for many living organisms to perform normal
physiological functions [2]. It is an integral part of many enzymes that perform critical physiological activities.
For example, mitochondrial electron transport and melanin production are highly regulated, with copper-based
enzymes playing an active part [3]. However, while copper is beneficial within certain concentrations, it is
considered toxic at higher concentrations [4]. Owing to its typical redox behavior, copper participates in
reactions that ultimately produce reactive oxygen species, leading to the deterioration of cellular machineries
including membranes and DNA components [5]. Furthermore, abnormal copper concentrations in plasma are
frequently observed under leukemic conditions [6].
Determination of halide ions is an important research topic for sustainable development because of their many
industrial applications, as well as their considerable environmental and biological impacts [7]. Bromide ions are
found in diverse sources, including fresh and marine water, and many biological fluids. Exposure to bromide
ions may cause various health problems including skin conditions, tissue damage, respiratory system
dysfunction, and cancer induction [8].
Various analytical methods have been developed for the detection of cations and anions [9]. These strategies
include designing organic molecules with cation binding affinity to form a receptor–cation complex, which
tends to interact with a particular anion, resulting in ion-pair recognition. Herein, upon metal binding, the
electronic distribution and structural aspects of the receptors are modified, which can be observed by
spectroscopic and conductometric techniques [10]. Fluorescence spectroscopic methods are frequently used
because they are fast, easy, and economical [11]. The recognition of anions typically depends on hydrogen
bonding, whereby there is competition between an anion and a polar solvent [12]. Ion-pair recognition provides
scope for additional electrostatic interactions, improving the binding affinity for the analyte.
2

Several tripodal receptor systems have been reported and fabricated with three arms to which ligating groups
were attached [13]. The selectivity of such tripodal receptors generally depends on the rigidity of their arms, the
cavity size, and the directional orientation of binding groups [14–16]. Compared with monopodal or dipodal
receptors, the typical structural features of tripodal receptors include their enhanced chelating effect, strong
binding affinity, and easy tunability towards a specific metal ion [17,18]. In this study, to take advantage of
these properties, a tripodal receptor was developed for the detection of Cu2+ and Br–. Only a few receptors have
been reported for the detection of Br–, due to its lack of suitable binding sites, low charge density, and weak
tendency to form hydrogen bonds [19–21]. Therefore, fast, reliable, and economical methods for the detection
of copper and bromide ions are in high demand.
2.
Results and Discussion
2.1
Synthesis of R1
Compound 1 was synthesized by the reaction of tris(2-chloroethyl)amine with salicylaldehyde in the presence
of NaOH under reflux [22]. R1 was obtained by a condensation of compound 1 with p-toluenesulfonyl
hydrazide in the presence of a catalytic amount of Zn(ClO4)2·6H2O at room temperature, and was characterized
by 1H and 13C NMR spectroscopy, IR spectroscopy, and ESI-MS and elemental analysis (Scheme 1).
3

Scheme 1. Synthesis of tripodal R1.
2.2
Cation Binding Studies
The UV-vis spectrum of R1 showed absorption bands at 270 and 315 nm. The fluorescence emission profile of
R1 was recorded with excitation at 270 nm, and exhibited an emission band at 415 nm. All spectroscopic
studies were performed in acetonitrile. UV-vis absorption and fluorescence spectroscopic methods were used to
examine the binding affinity of R1 towards various metal ions (Na+, K+, Ag+, Ba2+, Ca2+, Mg2+, Cd2+, Co2+,
Ni2+, and Zn2+) as nitrate salts. No major changes in the absorption spectra were observed in the presence of any
of the metal ions, as shown in Figure S1. On the other hand, the fluorescence spectrum of R1 showed enhanced
fluorescence intensity upon the addition of Cu2+. The intensity of the emission band at 415 nm decreased upon
the addition of Cu2+, while new emission bands at 310 and 446 nm were observed with 10-fold enhancement
(Figure 1 and S2), displaying the selectivity of R1 towards Cu2+. The fluorescence enhancement of R1 may be
attributed to chelation of R1 with Cu2+, which increases the rigidity of the molecular assembly, resulting in
chelation-enhanced fluorescence [23,24]. To determine the binding stoichiometry, Job’s plot was drawn (Figure
S3A) [25], revealing a 1:1 stoichiometry for the complex formed between R1 and Cu2+. The binding
stoichiometry of was further supported by ESI-mass spectrometry analysis. A peak at m/z = 1028.14 was
assigned to [R1 + Cu2+] (calcd. m/z = 1028.22) (Figure S3B). Upon the successive addition of Cu2+ (0–10
equiv.) to a 5 µM solution of R1, the emission bands at 310 and 446 nm gradually increased in intensity (Figure
2). A calibration curve for quantitative determination of Cu2+ with R1 was constructed, as shown in Figure 3.
4

R1 exhibited a good linear correlation for concentration (0–20 µM) vs. fluorescence intensity at 310 nm, with a
correlation coefficient R2 = 0.99, indicating that R1 is a suitable receptor for the quantitative estimation of Cu2+.
A Benesi–Hildebrand plot was used to determine the stability constant of Cu2+ binding, which was found to be
(1.4 0.2) × 104 M-1 (Figure S4) [26]. The detection limit of R1 for Cu2+ was found to be 4.60 µM [27], which
is significantly lower than the concentration recommended by the World Health Organization guideline (31.5
µM) for drinking water (Figure 4) [28]. This result indicates that R1 is sensitive enough to monitor the Cu2+
concentration in water. Response time is also an important factor in determining analytes. The response time of
R1 towards Cu2+ was measured by studying the emission profile at different time intervals, indicating that 180 s
was long enough to sense Cu2+ (Figure S5). The effect of pH on the fluorescence changes of R1 towards Cu2+
was investigated at different pH values. The optimum fluorescence behavior of R1 was observed at a range of
pH 6.5-8 (Figure S6). To evaluate the possible interference of other cations with the Cu2+ sensing ability of R1,
competitive binding studies were performed in the presence of potential interfering metal ions. The results are
presented in Figure 5. No significant changes in fluorescence intensity at 310 nm were observed, confirming
that R1 displayed good Cu2+ recognition ability even in the presence of other metal ions.
Figure 1. Fluorescence intensity of R1 in CH3CN (5 µM) upon addition of a metal salt (10 equiv.) excited at
270 nm.
5

Figure 2. Changes in fluorescence intensity of R1 (5 µM) upon successive additions of Cu2+ (0–10 equiv.).
Figure 3. Plot of fluorescence intensity at 310 nm as a function of the concentration of Cu2+.
6

Figure 4. Calculated detection limits for the estimation of Cu2+ by R1.
160
140
120
100
80
60
40
20
0
Figure 5. Fluorescence responses of R1 (5 µM) to Cu2+ (3 equiv.) in the presence of equivalent amounts of
other metal ions.
2.3
Anion Binding
To investigate the R1–Cu2+ complex as a secondary sensor for anion recognition, a solution of R1–Cu2+ was
prepared in situ by the addition of 10 equiv. of Cu2+ to an R1 solution (10 µM). The resulting solution was
7

–
–
–
–
screened with tetrabutylammonium salts of F–, Cl–, I–, H2PO4 , HSO4 , CN–, NO3 , OAc–, and ClO4 (Figure 6,
S7 and S8). None of the anions examined showed spectroscopic changes in the complex. However, upon
addition of Br–, the fluorescence intensity of R1–Cu2+ was quenched drastically at 310 and 446 nm. The
fluorescence intensity at 310 nm decreased to about 90% of that of R1–Cu2+. The shape of the fluorescence
spectrum at the final stage was similar to that at the beginning stage, indicating that R1 was released from the
complex by the binding of Cu2+ with Br–, rather than by forming a new complex. It is noteworthy that even after
adding 11 equiv. of Br–, the fluorescence intensity of the solution could not regain the initial emission state of
R1. This phenomenon might be attributed to the tight binding between R1 and Cu2+, resulting in incomplete
displacement of Cu2+ from the complex. Job’s plot analysis showed a 1:1 stoichiometry between R1–Cu2+ and
Br– (Figure S9). To investigate the binding properties of R1–Cu2+, titration experiments were performed by
successive additions of Br– (0–11 equiv.) while monitoring the emission spectra (Figure 7). A linear regression
in fluorescence intensity was observed in the range 0–50 µM, with a high correlation coefficient of R2 = 0.99
(Figure 8). This result shows that R1–Cu2+ can be used as a probe for quantitative estimation of Br–. The
association constant of R1–Cu2+ was measured to be (8.4 0.4) × 103 M−1 using the Benesi–Hildebrand
equation (Figure S10). The detection limit for Br– was calculated to be 9.33 µM (Figure 9). Competition
experiments were performed in the presence of other anions to evaluate their interference in estimating Br– with
R1–Cu2+ (Figure 10). When R1–Cu2+ was treated with 10 equiv. of Br– in the presence of equivalent amounts
of other anions, no significant interference was observed.
8

0
-0.2
-0.4
-0.6
-0.8
-1
Figure 6. Anion selectivity profiles of R1–Cu2+ (5 µM) in the presence of various anions (10 equiv.) in CH3CN
at 310 nm, excited at 270 nm.
Figure 7. Changes in fluorescence intensity of R1–Cu2+ (5 µM) upon addition of Br– (0–11 equiv.).
9

Figure 8. Fluorescence intensity vs. Br– concentration linear regression plot of R1–Cu2+ (5 µM) upon addition
of Br– at 310 nm.
Figure 9. Detection limit calculation of R1–Cu2+ towards Br–.
10

120
100
80
60
40
20
0
Figure 10. Interference of anions in the recognition of Br– by R1–Cu2+.
2.4 Sensing mechanism
To understand the binding mode of R1 with Cu2+, theoretical calculations were performed using DMoL 3 package.
The generalized gradient approximations (GGA) and double numeric plus polarization (DNP) was used [29].
The HOMO-LUMO energy gaps in R1 and R1–Cu2+ were calculated to be 1.172 eV and 0.441 eV, respectively,
showing that the binding of R1 with Cu2+ leads to decrease the HOMO-LUMO energy gap of the complex as
shown in Figure 11. These results rationalize the changes in fluorescence intensity of R1 upon Cu2+ addition. The
optimized structural features of R1–Cu2+ show that Cu2+ ion binds to R1 at oxygen atoms of the sulfonyl groups.
In addition, upon the addition of Br–, the shape of the fluorescence emission profile of R1–Cu2+ was unchanged
while the intensity of fluorescence decreased. These results indicate that the copper ion in R1–Cu2+ is released
from the complex upon the addition of Br– rather than forming a new complex. A plausible binding mode of R1
with Cu2+ and Br– is presented in Scheme 2.
11

Figure 11. Energy correlation of HOMO-LUMO gaps between R1 and R1–Cu2+ complex.
Scheme 2. Proposed mechanism of Br– detection by the R1–Cu2+ complex.
3.
Conclusions
We have synthesized a tripodal receptor R1, incorporating both nitrogen- and oxygen-containing binding sites,
and investigated its cation binding properties towards sensor development. Herein, R1 showed high selectivity
12

for Cu2+, with the emergence of new peaks as well as marked enhancement of fluorescence intensity upon
binding. The receptor was also found to recognize Cu2+ with a detection limit of 4.60 µM without interference
from other metal ions. The resulting R1–Cu2+ complex was further studied for its anion recognition ability. R1–
Cu2+ was found to be highly selective for Br–, showing a concentration-dependent behavior with no interference
from other anions. This sensing system may have applications in estimating biologically important Cu2+, as well
as Br–.
Supplementary data
Experimental details and NMR spectra associated with this article can be found, in the online version, at
https://doi.org/.
References
[1] Aragay G, Pons J, Merkoçi A. Chem Rev. 2011;111:3433–3458.
[2] Vassallo N, Herms J. J Neurochem. 2003;86:538–544.
[3] Massaro EJ. Handbook of Copper Pharmacology and Toxicology. 1st ed. New York: Springer-Verlag;
2002.
[4] Gaetke LM, Chow CK. Toxicology. 2003;189:147–163.
[5] Halliwell B, Gutteridge J. Biochem J. 1984;219:1–14.
[6] Lane E, Holden AJ, Coward RA. Analyst. 1999;124:245–249.
[7] Singh A, Singh J, Singh N, Jang DO. Tetrahedron. 2015;71:6143–6147.
13

[8] Yasri S, Wiwanitkit V. South Asian J Cancer. 2016;5:62.
[9] Singh A, Raj T, Aree T, Singh N. Inorg Chem. 2013;52:13830–13832.
[10]
[11]
[12]
[13]
[14]
[15]
Singh N, Jang DO. Org Lett. 2007;9:1991–1994.
Kaur N, Singh N, Cairns D, Callan JF. Org Lett. 2009;11:2229–2232.
Goh H, Nam TK, Singh A, Singh N, Jang DO. Tetrahedron Lett. 2017;58:1040–1045.
Kuswandi B, Verboom W, Reinhoudt DN. Sensors. 2006;6:978–1017.
Sato K, Arai S, Yamagishi T. Tetrahedron Lett. 1999;40:5219–22.
Ballester P, Costa A, Deyà PM, Vega M, Morey J, Deslongchamps G. Tetrahedron Lett.
1999;40:171–174.
[16]
[17]
[18]
Fan A, Hong HK, Valiyaveettil S, Vittal JJ. J Supramol Chem. 2002;2:247–54.
Berrocal MJ, Cruz A, Badr IH, Bachas LG. Anal Chem. 2000;72:5295–5299.
Reinoso‐García MM, Dijkman A, Verboom W, Reinhoudt DN, Malinowska E, Wojciechowska
D, Pietrzak M, Selucky P. Eur J Org Chem. 2005;2005:2131–2138.
[19]
Mahapatra AK, Roy J, Sahoo P, Mukhopadhyay SK, Chattopadhyay A. Org Biomol Chem.
2012;10:2231–2236.
[20]
[21]
[22]
[23]
Barman S, Sadhukhan M. Mater Chem. 2012;22:21832–1837.
Singh A, Singh A, Singh N, Jang DO. Tetrahedron. 2016;72:3535–3541.
Ragunathan KG, Bharadwaj PK. Tetrahedron Lett. 1992;33:7581–7584.
Oliveira E, Santos HM, Capelo JL, Lodeiro C. Inorg Chim Acta. 2012;381:203–211.
14

[24]
Li W, Tian XH, Huang B, Li HJ, Zhao XY, Gao S, Zheng J, Zhang XZ, Zhou HP, Tian YP, Wu
JY. Biosens Bioelectron. 2016;77:530–536.
[25]
Patrick M, Hill ZD. Chem Educ. 1986;63:162–167.
[26]
[27]
[28]
Benesi H, Hildebrand HJ. J Am Chem Soc. 1949;71:2703–2707.
Long GL, Winefordner JD. Anal Chem. 1983;55:712A–724A.
Dean JA. Langès Handbook of Chemistry. 15th ed. New York: McGraw-Hill; 1999.
Tao JM, Perdew JP, Staroverov VN, Scuseria GE. Phys Rev Lett. 2003;91:146401–146404.
[29]
Highlights
ꢀ A tripodal receptor R1 was used to explore the selective determination of Cu2+.
ꢀ R1 in the presence of Cu2+ showed a fluorescence turn-on.
ꢀ R1–Cu2+ complex showed the selectivity for Br– with a fluorescence turn-off.
[30]
Graphical Abstract
Single chemosensor for sensing multiple analytes: selective fluorogenic detection of Cu2+ and Br–
Yoon Gun Ko, Mayank, Narinder Singh* and Doo Ok Jang*
15

[31]
16