A uracil nitroso amine based colorimetric sensor for the detection of Cu2+ ions from aqueous environment and its practical applications

Article abstract of DOI:10.1039/c4ra10419f

A simple uracil nitroso amine based colorimetric chemosensor (UNA-1) has been synthesized and screened for its cation recognition ability. Sensor UNA-1 exhibited a high sensitivity and selectivity towards Cu²⁺ ions in aqueous medium in the presence of a wide range of other competing cations (Ag⁺, Al³⁺, Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cs⁺, Fe²⁺, Fe³⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, Zn²⁺, Hg²⁺ and Sr²⁺). With Cu²⁺, the sensor UNA-1 gave a distinct color change from colorless to dark yellow by forming a complex of 1:1 stoichiometry. Furthermore, sensor UNA-1 was successfully utilized in the preparation of test strips and supported silica for the detection of Cu²⁺ ions from aqueous environment.

Full text of DOI:10.1039/c4ra10419f

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Table of contents
Uracil nitroso amine based colorimetric chemosensor for Cu2+ ions from a 100 % aqueous
environment

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A Uracil nitroso amine based colorimetric sensor for the detection of Cu2+ ions from
aqueous environment and its practical applications
Samadhan R. Patila, Jitendra P. Nandrea, Prashant A. Patilb, Suban K. Sahooc, Manisha Devid,
Chullikkattil P. Pradeepd, Yu Fabiaoe, Lingxin Chene, Carl Redshawf, Umesh D. Patila*
a School of Chemical Sciences, North Maharashtra University, Jalgaonꢀ425 001, (M.S.) India,
Mob: +91ꢀ9273060210; Eꢀmail: udpatil.nmu@gmail.com (UDP, former Assi. Prof.).
b S.S.V.P.S’s L. K. Dr. P. R. Ghogrey Science College, Dhuleꢀ424 001, India.
c
Department of Applied Chemistry, S. V. National Institute Technology, Suratꢀ395 007,
Gujrat, India.
d
School of Basic Sciences, Indian Institute of Technology, Mandi, Himachal Pradeshꢀ
175001, India.
e
Key Laboratory of Coastal Zone Environmental Processes and Ecological Remediation,
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003,
China.
f Department of Chemistry, University of Hull, Cottingham Road, Hull, HU6 7RX (UK).
Abstract
A simple uracil nitroso amine based colorimetric chemosensor (UNA-1) has been
synthesized and screened for its cation recognition ability. Sensor UNA-1 exhibited a high
sensitivity and selectivity towards Cu2+ ions in aqueous medium in the presence of a wide
range of other competing cations (Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cs+, Fe2+, Fe3+, Li+,
Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+, Hg2+ and Sr2+). With Cu2+, the sensor UNA-1 gave a
distinct color change from colorless to dark yellow by forming a complex of 1:1
stoichiometry. Furthermore, sensor UNA-1 was successfully utilized in the preparation of test
strips and supported silica for the detection of Cu2+ ions from aqueous environment.
Key words: Colorimetric chemosensor; Cu2+; Uracil nitroso amine, aqueous environment;
DFT.
1

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Introduction
Copper, the thirdꢀmost abundant transition element in the body, plays an important
role in various physiological processes such as hemoglobin biosynthesis, bone development,
dopamine production, nerve function regulation, gene expression, and the functional and
structural enhancement of proteins.1ꢀ6 Due to its redoxꢀactive nature, copper serves as an
essential coꢀfactor for a variety of metalloenzymes in living organisms such as cytochrome c
oxidase, lysyl oxidase, copperꢀzinc superoxide dismutase and tyrosinase, which have a
significant role in the enzymatic defense against oxygen toxicity.7ꢀ9 However, at high
concentration, its redox properties turn into biologically hazardous materials because of its
ability to generate reactive species, which create problems in the cellular metabolism.8,9 Apart
from the biological and environmental importance, other advantages are that copper is
relatively abundant, of low cost, and possess good malleability, electrical conductivity,
thermal conductivity, chemical stability as well as germicidal efficiency. All of these
properties make copper central to the pharmaceutical and industrial sectors for making alloys,
electrical wires, machine parts, batteries, drugs and fertilizers etc.10,11 However, with
excessive loading, Cu2+ is highly toxic to living organisms. For example, it’s over
accumulation in human being leads to various diseases including neurodegenerative diseases
such as Alzheimer’s disease, Wilson’s disease, Menkes disease, prion disease,
gastrointestinal disorders, kidney damage, amyotrophic sclerosis, lipid metabolism and
inflammatory disorders.12ꢀ15
The World Health Organization (WHO) have reported that the maximum limit of
copper in drinking water should be 2 ppm (30 µM).16 Under normal conditions, the average
concentration of copper in the blood should not exceed 100ꢀ150 µg/dL.17 However, due to the
widespread use of copper in household appliances, industry, agricultural and waterꢀpipes,
Cu2+ pollution has increased immensely throughout the world. Therefore, it is necessary to
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develop fast, convenient and reliable analytical methods for the qualitative and quantitative
detection of copper, particularly in drinking water and in biological samples. Several
analytical techniques such as atomic absorption spectrometry (AAS), inductively coupled
plasma mass spectroscopy (ICPꢀMS), inductively coupled plasma atomic emission
spectrometry (ICPꢀAES) and voltammetry, quantumꢀdotꢀbased assay have been developed
for the qualitative and quantitative detection of Cu2+ ions at trace levels. These technologies
can detect Cu2+ ion selectively with high sensitivity, but tend to need highly sophisticated and
expensive instrumentation, and require tedious sample preparation and highly trained
operators.18ꢀ21 By contrast, nakedꢀeye detection methods permit detection of the target analyte
at the micro/submicromolar levels without any need for expensive/sophisticated
instrumentation.22,23 Therefore, given the importance of and the hazardous roles played by
copper, we were encouraged to develop a colorimetric sensor with nakedꢀeye capability for
detecting Cu2+ from pure aqueous media.
On surveying the literature, we have noted that most of the reported Cu2+ selective
colorimetric sensors have a number of drawbacks, viz long response times, poor detection
limits, tedious synthetic procedures, use of organic solvents, and interference from other
transition metal ions (Table S1).22ꢀ32 Herein, as a part of our ongoing research on
chemosensors,32ꢀ35 we have developed a simple and easy to prepare colorimetric
chemosensor, namely the uracil nitroso amine derivative UNA-1 (Scheme 1), which can be
used for the highly selective and sensitive recognition of Cu2+ ions. This chemosensor gives a
visual color change from colorless to clear dark yellow allowing for the nakedꢀeye detection
of Cu2+.
Results and Discussion
Naked-eye detection of Cu2+
The synthesis of the uracil nitroso amine derivative (UNA-1) was achieved in two
steps by the reaction of ethylꢀcyanoacetate and Nꢀmethylurea in the presence of sodium
3

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ethoxide as a catalyst under acidic medium to give pyrimidine 1, which further undergoes
nitrosation with sodium nitrite to afford UNA-1 (Scheme 1). Then, the colorimetric sensing
ability of UNA-1 (5 х 10ꢀ5 M, in CH3OH:H2O, 10:90, v/v) was tested via the addition of 5
equivalents of various cations (1 х 10ꢀ2 M, in H2O). As shown in Fig. 1, UNA-1 exhibits a
selective, sensitive and qualitative recognition of Cu2+ ions in day light/sunlight through a
distinct visual color change from colorless to dark yellow. No noticeable color change was
observed in the presence of any other cations screened herein.
Scheme 1. Synthesis of UNA-1
Fig. 1. Nakedꢀeye detectable color change of ions with UNA-1 in the presence of 5
equivalents of different cations.
The concentration dependent nakedꢀeye study was performed (Fig. S1a) by addition
of various concentrations of UNA-1 (A =1 х 10ꢀ3 M, B = 1 х 10ꢀ4 M, C = 1 х 10ꢀ5 M, D = 5 х
10ꢀ5, E = 1 х 10ꢀ6 M, F = 5 х 10ꢀ6 M and G = 1 х 10ꢀ7 M) to a fixed concentration of Cu2+ ions
(1 х 10ꢀ3 M, in H2O). The observed color change clearly suggested that the sensor was quite
sensitive up to a concentration of 5 х 10ꢀ5 M for the detection of Cu2+ ions. Next, we
investigated the effect of changing the concentration of Cu2+ ions from 1 х 10ꢀ3 M to 1 х 10ꢀ7
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M to a fixed concentration of UNA-1 (1 х 10ꢀ4 M), which inferred that our sensor was able to
detect Cu2+ up to the concentration of 1 х 10ꢀ5 M (Fig. S1b).
Cation sensing studies
The cation recognition behavior of sensor UNA-1 with group I, II and III metal ions
(Ba2+, Ca2+, Cs+, Li+, Mg2+, Na+, Al3+ and Sr2+) and transition and heavy metal ions (Co2+,
Cu2+, Cr3+, Fe2+, Cd2+, Fe3+, Mn2+, Ni2+, Pb2+, Zn2+ and Hg2+) was investigated using UVꢀVis
absorption spectroscopy. The absorption spectrum of sensor UNA-1 in CH3OH:H2O (10:90,
v/v) solvent system exhibited two absorption bands centered at 227 nm and 315 nm due to πꢀ
π* and nꢀπ* electronic transitions, respectively. Upon addition of 5 equivalents of different
cations (50 µL, 1 х 10ꢀ2 M, in water) to a 5 х 10ꢀ5 M solution of UNA-1 in CH3OH:H2O
(10:90, v/v), only the Cu2+ ions was able to perturb the absorption spectrum of UNA-1
effectively. The addition of aqueous Cu2+ ions to the UNA-1 solution led to the disappearance
of the absorption band at 315 nm and the appearance of a new broad band between 335ꢀ500
nm due to the interaction of the paramagnetic Cu2+ with UNA-1 (Fig. 2). The appearance of a
new charge transfer band was responsible for the nakedꢀeye detectable color change of UNA-
1.
The UVꢀVis absorption titration was next performed upon successive addition of 1ꢀ10
equivalents of Cu2+ ions to the solution of UNA-1 to determine the binding ability and the
limit of detection. With the incremental addition of Cu2+, the absorbance at wavelength 315
nm decreased continuously with the appearance of the new broad peak between 335ꢀ500 nm
(Fig. 3). The titration resulted in the formation of an isosbestic point at 300 nm, which
suggested the formation of a complex between UNA-1 and copper ions in solution.
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Fig. 2. Absorption spectra of UNA-1 in the presence of 5 equivalents of different cation.
Fig. 3. Changes in absorption spectra of UNA-1 upon incremental addition of Cu2+ ions (1ꢀ10
equiv.). Inset showing mole ratio plot i.e. change in absorption spectra (∆A at λmax=315 nm)
as a function of concentration of Cu2+ ions.
The association constant (Ka) was estimated graphically by plotting 1/∆A against 1/
[Cu2+] (Fig. 4). The data was linear (fitted according to the Benesi–Hilderbrand equation) and
the Ka value was obtained from the slope and intercept of the line. The Ka value for the UNA-
1 copper complex was found to be 2.8 × 104 Mꢀ1 (R2=0.9933). The value suggested that the
sensor UNA-1 has high affinity towards Cu2+ ions. The limit of detection (LOD) and limit of
quantification (LOQ) of UNA-1 were also calculated from the absorption titration data.
According to the IUPAC definition, the LOD and LOQ were calculated using the relationship
LOD = (3.3 x standard deviation)/slope and LOQ = (10 x standard deviation)/slope. To
calculate the relative standard deviation, the absorption measurements of ten blank samples
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were taken. As shown in Fig. S2, the absorbance calibration values were normalized between
the minimum intensity and the maximum intensity and then a linear regression curve was
fitted to these normalized data to get the slope. With this approach, the LOD and LOQ were
found to be 10 µM and 33 µM, respectively.
Fig. 4. Benesi–Hilderbrand plot of chemosensor UNA-1 with Cu2+ ion for evaluation of
association constant or binding constant (where, ∆A is at λmax=315 nm).
The 1:1 binding stoichiometry for the complexation between UNA-1 and Cu2+ was
determined using a Job’s plot experiment (Fig. 5) and a mole ratio plot (Fig. 3, inset).
Furthermore, more direct evidence for the formation of this 1:1 complex was obtained from
the ESIꢀMS spectra of UNA-1 in the presence of 1.0 equivalent of Cu2+ in methanol/water
(10:90, v/v) (Fig. S3). For pure UNA-1, a characteristic peak at m/z = 207.0405 was obtained
which corresponds to the species [(UNA-1).2H2O+H], whilst on addition of copper
perchlorate, the peak at 207.0405 disappeared and a new peak appeared at m/z = 287.0051
corresponding to the species [(UNA-1ꢀH).Cu.3H2O]+ (Fig. S3). MSꢀMS of 287.0051 peak
corresponding to the hydrated copper complex of UNA-1 shows fragmentation giving peaks
at 251.94 and 233.97 corresponding to the species [(UNA-1)-H.Cu.H2O]+ and [(UNA-1)-
H.Cu]+ respectively (Fig. S3).
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Fig. 5. Job’s plot for the determination of a 1:1 stoichiometry upon complexation of UNA-1
with Cu2+ ions (where, ∆A is at λmax=315 nm).
The chemosensor UNA-1 can bind to the Cu2+ ion via binding sites consisting of an
amino and a nitroso group. All the crystal structures reported for complexes with similar
ligands show the coordination through amino nitrogen and nitrogen of nitroso group.36ꢀ41
Thus, the lone pair of electrons on the nitrogen atoms of the amino and nitroso groups of the
sensor UNA-1 are delocalized to the vacant orbital localized on the Cu2+ as shown in the Fig.
6. This electron donation or charge transfer gave rise to a color change from colorless to clear
yellow. The charge of the copper is +2 and hence there should be two negative charges in our
proposed structure for charge neutrality. Therefore, we propose the deprotonation of ꢀNH2
ꢀ
group and the inclusion of ClO4 counter ion in the complex formula. Further, for more
1
evidence of the binding of the Cu2+, we carried out H NMR titration studies on UNA-1 by
adding Cu2+ solutions (Fig. S4). It was observed that the peak at δ 12.91 corresponding to the
–NH2 protons showed an upꢀfield shift from 12.91 to 12.52 ppm accompanied by a
broadening of the peak, while the peak at δ 9.08 corresponding to the –NH proton shows a
small downfield shift from 9.08 to 9.28 ppm with broadening of the peak on addition of 1.0
equivalent of copper perchlorate. These observed shifts could be due to the complexation as
proposed earlier. The possible 3D structure and the charge transfer processes occurring
during the encapsulation of Cu2+ by UNA-1 was investigated by density functional theory
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(DFT) calculations. The optimized structure of UNA-1 and its complex with Cu2+ are shown
in Fig. 7. On complexation of UNA-1 with Cu2+, a lowering in the interaction energy by ꢀ
145.16 kcal/mol was observed, which indicates the formation of a stable complex with the
calculated average CuꢀN bond length of 2.062 Å. Further, the analysis of the frontier
molecular orbitals (FMOs) plots (Fig. 7b-c) of the UNA-1.Cu2+ complex indicates that the
intramolecular charge transfer (ICT) occurred between the receptor UNA-1 and Cu2+. Also,
the band gap between the beta HOMO and LUMO of UNA-1.Cu2+ complex was lowered
than the receptor UNA-1, which caused the experimentally observed redꢀshift in the
absorption band.
Fig. 6. Proposed mode of binding of Cu2+ ion with UNA-1 as shown by the color change.
Fig. 7. DFT computed (a) optimized structure of UNA-1 and its complex with Cu2+, and the
(b) LUMO and (c) HOMO diagrams of UNA-1 and its UNA-1).Cu2+ complex.
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Practical applications of UNA-1
The analytical applicability of UNA-1 was first tested by performing competitive
experiments. The absorption and color changes caused by the mixture of Cu2+ with the other
metal ions was similar to that caused by Cu2+ alone (Fig. S5), which indicates that the other
metal ions did not interfere with the binding of the chemosensor UNA-1 with Cu2+. Secondly,
the reversibility of UNA-1 for the detection of Cu2+ was examined. To perform the
reversibility test, a stock solution of UNA-1 (1 х 10ꢀ3 M) was first treated with 1 equivalent of
Cu2+. The color of the solution changes from colorless to yellow. To the same solution, the
reverse color change from yellow to colorless was observed upon addition of 4 equivalents of
aqueous EDTA solution (Fig. S6 and Supporting video). This result demonstrated the
reversibility of the sensor UNA-1.
The sensing of Cu2+ by UNA-1 worked very well on a solid support (Fig. 8 and
supporting video). In this experiment, the silica gel (60–120 mesh, 10.0 g, colorless) was
soaked with UNA-1 (in methanol, 50 mL, 1х10−2 M) and then dried to afford a faint pink
color silica gel due to the adsorption of the sensor on the surface. When the treated silica gel
was added to a 10 mL aqueous solution of Cu2+ (1х10−3 M), the faint pink color promptly
turned to a dark greenish/yellow color (supporting video). The instantaneous color change of
the solid silica gel in aqueous solution clearly inferred the practical application of UNA-1 for
the qualitative detection of Cu2+ in aqueous medium. Then, the UNA-1 supported silica gel
was treated with different concentrations of Cu2+ (B = 1 х 10−3 M, C = 1 × 10ꢀ4 M, D = 1 х
10−5 M, E = 1 х 10−6 M), which indicated that the silica gel can be used to detect Cu2+ up to 1
х 10−5 M by a visually detectable color change (Fig. 9). The results indicate that we can use
this silica supported method not only in the determination of Cu2+ ions from water but also in
the extraction / seperation of Cu2+ ions from water.
In another approach, the practical utility of UNA-1 for the detection of Cu2+ was
studied by developing a test paper strip. The cellulose paper (Whatman No. 42) was dipped in
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the methanolic solution of UNA-1 (1х10ꢀ2 M) followed by drying in air to prepare the desire
test strip. When this strip was dipped into an aqueous solution of Cu2+ (1х10−3 M), the
colorless strip sharply turned to a yellow color (Fig. S7 and supporting video). The rapid
color change of the test strip in solution clearly inferred the practical application of UNA-1
for the qualitative detection of Cu2+ in aqueous medium.
Fig. 8. Application of sensor UNA-1 on supported silica, and color changes of silica gel with
/ without UNA-1 and Cu2+ solution (before and after the addition).
Fig. 9. UNA-1 supported silica gel was treated with different concentrations of Cu2+. A=silica
without UNA-1 + 1 х 10−3 M Cu2+ as controle.
Conclusion
In summary, we have developed a simple uracil nitroso amine derivative UNA-1 for
the colorimetric detection of Cu2+ in aqueous media. The sensor exhibited excellent
selectivity and high sensitivity towards Cu2+ ions. The recognition of Cu2+ induced a clearly
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distinct color change of UNA-1 from colorless to yellow allowing for nakedꢀeye detection.
Moreover, UNA-1 can be applied to the detection of Cu2+ in aqueous media by a test paper
strip and silica support method. These methods offer a very simple and quick detection for
Cu2+ in aqueous media with a detection limit down to 10 µM. The good selectivity of sensor
UNA-1 towards Cu2+ coupled with the use of pure water as the sole solvent in the detection
process makes UNA-1 a promising candidate for the qualitative and quantitative detection of
Cu2+ in various chemical, environmental and biological systems.
Experimental
Chemicals and Instrumentations
Unless otherwise stated, all chemicals used for the synthesis of UNA-1 were of AR
grade and were purchased either from S.D. Fine chemicals or Sigma Aldrich. All solvents
were of spectroscopic grade and were used without further treatment. The aqueous stock
solutions of cations (1 х 10ꢀ2 M) such as Ag+, Al3+, Ba2+, Cd2+, Co2+, Cu2+, Cr3+, Fe2+, Fe3+,
Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+ were prepared from their perchlorates salts; Ca2+, Na+, Sr2+
and Cs+ were prepared from their nitrate salts; Hg2+ from its chloride salt and Li+ from its
bromide salt. The stock solution of UNA-1 (1 х 10ꢀ3 M) was prepared in methanol and then
diluted to 5 х 10ꢀ5 molLꢀ1 with methanol:water (10:90, v/v).
1
The H and 13C NMR spectra were recorded on a Jeol JNMꢀECX 500 MHz
multinuclear probe NMR spectrometer at ambient temperature in DMSOꢀd6 with TMS as
internal standard and chemical shifts reported in ppm. Mass spectra were recorded on a
Bruker Compact HD mass spectrometer. The IR spectra were recorded on a Perkin Elmer
FTIR spectrophotometer by using KBr discs and the IR bands are expressed in frequency
(cmꢀ1). Absorption spectra were recorded on a Perkin Elmer U 3900 Co, USA UV/Visible
double beam spectrophotometer. The purity of the compound and progress of the reaction
was monitored by means of a thin layer chromatography (TLC). Preꢀcoated silica gel 60 F254
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(Merck) on alumina plate (7 х 3 cm2) was used and visualized by using either an iodine or a
short UV/Visible lamp. Melting points were recorded on the Celsius scale by open capillary
method and are uncorrected.
Synthesis of UNA-1
The chemosensor UNA-1 was synthesized by following the reported method42 in two
steps as depicted in Scheme 1. In the first step, ethyl cyanoacetate (2.1 mL, 0.019 mol) was
added from a dropping funnel under vigorous stirring to a solution of sodium ethoxide
prepared from 0.92 g of metallic sodium and 20 mL of ethanol. A white solid was separated
from the solution. After the complete addition of ethyl cyanoacetate, the mixture was stirred
for 20 min at room temperature. Nꢀmethylurea (1.20 g, 0.016 mol) was added and the mixture
was heated for 3 h under reflux conditions on a water bath. The obtained white precipitate
was filtered off, washed with ethanol, and then dissolved in 10 mL of water. The pH of the
solution was adjusted to 7 by adding dilute sulphuric acid (2 N), and the mixture was stirred
for 2 h to afford the pyrimidine 1 as product in 95 % yield, m.p > 300 0C.
In the second step, a solution of 1.50 g of sodium nitrite in 4.0 mL of water was added
to a mixture of 2.41 g of 5ꢀaminoꢀ2,4ꢀdihydroxypyrimidine (1) and 12 mL of water. Then,
1.70 g of conc. H2SO4 was added dropwise under vigorous stirring. A solid precipitated,
which was stirred for 6 h at room temperature, and the obtained product was filtered and
washed with ethanol and water. Yield 86 %. IR spectrum, ν, cmꢀ1: 3550, 3320, 3157, 3040,
2968, 2851, 1722, 1712, 1666, 1630, 1513, 1594, 1513, 1462, 1436, 1385, 1288, 1237, 1140,
1079, 1053, 512, 491. HRMS: m/z 207.0405 corresponding to the species (UNAꢀ1).2H2O+H.
1H NMR (500 MHz, DMSO d6, δ ppm): 12.91 (s, 2H, ꢀNH2), 9.08 (s, 1H, ꢀNH), 3.25 (s, 3H,
ꢀCH3).13CꢀNMR (125 MHz, DMSO d6, δ ppm): 27.95 (ꢀCH3), 139.23 (ꢀCN=O), 146.25 (Cꢀ
NH2), 149.50 (C=O), 160.40 (C=O). (Fig. S8ꢀS11).
Computational study
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The structural optimization of UNA-1 and its hostꢀguest complexes with Cu2+ was
performed using the computer program Gaussian 09W43 by applying the density functional
theory (DFT) method. All the DFT calculations were performed in the gas phase with a
hybrid functional B3LYP (Becke’s three parameter hybrid functional using the LYP
correlation functional) using the basis sets 6ꢀ31G (d,p) for C, H, N, O atoms and LANL2DZ
for Cu atom.
Acknowledgement
S.R. Patil is thankful to DST, New Delhi, India, for financial assistant under INSPIRE
fellowship scheme and Dr. U. D. Patil is grateful for the financial support from the DST, New
Delhi, INDIA, under FAST TRACK scheme for Young Scientists, (Reg. No. CSꢀ088/2013).
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References
1
E. Gaggelli, H. Kozlowski, D. Valensin and G. Valensin, Chem. Rev. 2006, 106,
1995.
2
E. Crabb, E. Moore, L. E. Smart, Concepts in Transition Metal Chemistry, 1st ed.,
RSC Publishing, Cambridge, UK, 2010.
3
4
5
6
E. D. Harris, J. Trace Elem. Exp. Med., 2001, 14, 207.
C. Andreini, L. Banci, I. Bertini and A. Rosato, J. Proteome Res., 2008, 7, 209.
E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008, 108, 1517.
Z. Xu, K.ꢀH. Baek, H. Kim, J. Cui, X. Qian, D. R. Spring, I. Shin and J. Yoon, J. Am.
Chem. Soc., 2010, 132, 601.
7
K. D. Karlin, Science, 1993, 261, 701.
8
B. Halliwell and J. M. C. Gutteridge, Biochem. J., 1984, 219, 1.
M. C. Linder and M. HazeghꢀAzam, Am. J. Clin. Nutr., 1996, 63, 797S.
A. K. Jain, R. K. Singh, S. Jain and J. Raisoni, Transition Met. Chem., 2008, 33, 243.
P. G. Georgopoulos, A. Roy, M. J. YononeꢀLioy, R. E. Opiekun and P. J. Lioy, J.
Toxicol. Environ. Health B, 2001, 4, 341.
9
10
11
12
13
14
D. Strausak, J. F. B. Mercer, H. H. Dieter, W. Stremmel, G. Multhaup, Brain Res.
Bull., 2001, 55, 175.
E. Gaggelli, H. Kozlowski, D. Valensin and G. Valensin, Chem. Rev., 2006, 106,
1995.
G. Muthaup, A. Schlicksupp, L. Hess, D. Beher, T. Ruppert, C. L. Masters and K.
Beyreuther, Science, 1996, 271, 1406.
15
16
R. A. Løvstad, BioMetals, 2004, 17, 111.
WHO, WHO Guidelines Values for Chemicals that are of Health Significance in
Drinking Water. Guidelines for Drinking Water Quality, Geneva, 3rd edn, 2008.
15

Page 17 of 19
RSC Advances
View Article Online
DOI: 10.1039/C4RA10419F
17
18
19
R. B. Jonas, Appl. Environ. Microbiol., 1989, 55, 43.
N. Pourreza and R. Hoveizavi, Anal. Chim. Acta, 2005, 549, 124.
J. Becker, S. Zoriy, M. V. Pickhardt, C. N. PalomeroꢀGallagher and K. Zilles, Anal.
Chem., 2005, 77, 3208.
20
21
Y. Liu, P. Liang and L. Guo, Talanta, 2005, 68, 25.
J. OteroꢀRomani, A. MoredaꢀPineiro, A. BermejoꢀBarrera and P. BermejoꢀBarrera,
Anal. Chim. Acta, 2005, 536, 213.
22
23
X. Ma, Z. Tan, G. Wei, D. Wei and Y. Du, Analyst, 2012, 137, 1436.
Z. Xu, L. Zhang, R. Guo, T. Xiang, C. Wu, Z. Zheng and F. Yang, Sens. Actuators B,
2011, 156, 546.
24
X. Chen, M. J. Jou, H. Lee, S. Kou, J. Lim, S.ꢀW. Nam, S. Parka, K.ꢀM. Kim and J.
Yoon, Sens. Actuators B, 2009, 137, 597.
25
26
27
28
T. Gunnlaugsson, J. P. Leonard and N. S. Murray, Org. Lett., 2004, 6, 1557.
B. N. Ahamed and P. Ghosh, Dalton Trans., 2011, 40, 6411.
E. Hrishikesan, Saravanan and P. Kannan, Ind. Eng. Chem. Res., 2011, 50, 8225.
Y. Liu, Y. Sun, J. Du, X. Lv, Y. Zhao, M. Chen, P. Wang and W. Guo, Org. Biomol.
Chem., 2011, 9, 432.
29
30
R. Sheng, P. Wang, Y. Gao, Y. Wu, W. Liu, J. Ma, H. Li and S. Wu, Org. Lett., 2008,
10, 5015.
H. S. Jung, P. S. Kwon, J. W. Lee, J. Kim, C. S. Hong, J. W. Kim, S. Yan, J. Y. Lee,
J. H. Lee and T. Joo, J. Am. Chem. Soc., 2009, 131, 2008.
C. Zong, K. Ai, G. Zhang, H. Li and L. Lu, Anal. Chem., 2011, 83, 3126.
S. R. Patil, J. P. Nandre, D. Jadhav, S. Bothra, S. K. Sahoo, M. Devi, C. P. Pradeep, P.
P. Mahulikar, U. D. Patil, Dalton Trans., 2014, 43, 13299.
J. P. Nandre, S. R. Patil, V. S. Patil, F. Yu, L. Chen, S. K. Sahoo, T. Prior, C.
31
32
33
Redshaw, P. P. Mahulikar and U. D. Patil, Biosens. Bioelectron., 2014, 61, 612.
16

RSC Advances
Page 18 of 19
View Article Online
DOI: 10.1039/C4RA10419F
34
S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and J. F. Callan, Chem. Soc. Rev.,
2012, 41, 7195.
35
36
A. K. Gupta, A. Dhir and C. P. Pradeep, Dalton Trans., 2013, 42, 12819.
R. Kivekas, A. Pajunen, E. Colacio, J. M. DominguezꢀVera, J. M. Moreno and A.
Romerosa, Acta Chemica Scandinavica, 1997, 51, 1051.
37
38
39
40
41
E. Colacio, J. M. DominguezꢀVera, A. Escuer, R. Kivekas and A. Romerosa, Inorg.
Chem., 1994, 33, 3914.
E. Colacio, J. M. DominguezꢀVera, A. Romerosa, R. Kivekas, M. Klinga and A.
Escuer, Inorganica Chimica Acta, 1995, 234, 61.
G. Ferguson, J. N. Low, M. Q. Olozabal, J. M. S. Peregrin, F. H. Urena and M. N. M.
Carretero, Polyhedron, 1996, 15, 3233.
E. Colacio, J. M. DominguezꢀVera, A. Escuer, M. Klinga, R. Kivekas and A.
Romerosa, J. Chem. Soc., Dalton Trans., 1995, 343.
J. M. Salas, M. A. Romero, M. P. Sanchez, M. N. Moreno and M. Q. M. Faure,
Polyhedron, 1992, 11, 2217.
42
43
I. Fatima, M. A. Munawar and A. Tasneem, J. Mex. Chem. Soc., 2010, 54, 227.
A. H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V.
Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P.
Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M.
Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M.
Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C.
Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.
Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski,
17

Page 19 of 19
RSC Advances
View Article Online
DOI: 10.1039/C4RA10419F
G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J.
B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09, Revision A.1,
Gaussian, Inc., Wallingford CT (2009).
18