Novel coumarin-based fluorescent probe for selective detection of Cu(II)

Article abstract of DOI:10.1016/j.saa.2015.07.029

Abstract We report an efficient and convenient method for preparing nitro-3-carboxamide coumarin derivatives, proposed as novel fluorescent chemosensor, through microwave irradiation. This compound can be used as fluorescent probe for Cu²⁺ with selectivity over other metal ions in aqueous solution. The fluorescence of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide(3) is the highest in the presence of Cu²⁺, with stronger excitation at λ = 320 nm than for the other cations tested. copy; 2015 Elsevier B.V.

Full text of DOI:10.1016/j.saa.2015.07.029

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel coumarin-based fluorescent probe for selective detection of Cu(II)
⇑
Ahmadreza Bekhradnia , Elham Domehri, Masome Khosravi
Pharmaceutical Sciences Research Center, Department of Medicinal Chemistry, Mazandaran University of Medical Sciences, Sari, Iran
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
A new fluorescent chemosensor for
Cu has been reported.
We designed and synthesized a new
carboxamide coumarin sensor
through microwave irradiation.
O
2
+
O
O
O N
2
N
H
O N
2
N
Cu(II)
r.t
O
O
N(Me)2
Cu
O
N(Me)2
ꢀ
The association constant between 3
and Cu2+ was found to be
Fluorescence
Fluorescence
4
ꢂ1
3
.01 ꢁ 10 M
.
OFF
ON
a r t i c l e i n f o
a b s t r a c t
Article history:
We report an efficient and convenient method for preparing nitro-3-carboxamide coumarin derivatives,
proposed as novel fluorescent chemosensor, through microwave irradiation. This compound can be used
as fluorescent probe for Cu with selectivity over other metal ions in aqueous solution. The fluorescence
Received 6 February 2015
Received in revised form 24 June 2015
Accepted 6 July 2015
2+
of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide(3) is the highest in the pres-
Available online 7 July 2015
2
+
ence of Cu , with stronger excitation at k = 320 nm than for the other cations tested.
Ó 2015 Elsevier B.V. All rights reserved.
Keywords:
Cation recognition
Fluorescent chemosensor
Coumarin derivatives
1
. Introduction
the bloodstream and is deposited in the kidneys, cornea, and par-
ticularly, the brain [7c]. Therefore, detection and elimination of this
metal is important. In this regard, many analytical techniques are
available for the determination of heavy metals. Among them, sub-
stantial attention has been paid to fluorometry because of its high
sensitivity. Fluorescence can vary with polarity and viscosity
because it is sensitive to the local environment [6]. Coumarin is a
suitable fluorophore because of its desirable photophysical proper-
ties such as its large Stokes shift and visible excitation and emis-
sion wavelengths [8].
There are several methods for synthesizing substituted coumar-
ins, including the Pechmann and Duisberg [9], Perkin and Henry
[10], Brafola et al. [11], Claisen et al. [12], Shringer [13], and
Wittig reactions [14]. Because of the formation of chromone
by-products and variable yields, the synthesis of coumarins
through the Kostanecki–Robinson reaction is considered an infe-
rior procedure. Other approaches to coumarin preparation have
additional challenges. Most of these procedures must be carried
under severe conditions such as high temperatures, require long
reactions times, and afford low yields [15]. To overcome these
Coumarin derivatives are an important group of fluorescent
organic heterocycles with photochemical, photophysical, and bio-
logical properties that have led to variety of applications as phar-
maceuticals [1,2], fluorescent whiteners [3], fluorescent
indicators [4], and laser dyes [5]. Coumarin derivatives can be used
as fluorescent probes for metal ions because of their highly vari-
able size, hydrophobicity, and chelation 6. Selective detection of
heavy metal ions such as copper [7a] is important because of its
toxic and carcinogenic effects on humans and wildlife. For exam-
ple, the average diet provides substantial amounts of copper, and
the recommended intake is 0.9 mg/day [7b,c]. However, exposure
to higher levels of copper can cause rheumatoid arthritis, gastroin-
testinal disturbances, and Wilson’s disease. Copper is released into
⇑
Corresponding author at: Department of Chemistry and Molecular Biology,
University of Gothenburg, Gothenburg, Sweden.
E-mail addresses: abekhradnia@gmail.com,
A. Bekhradnia).
abekhradnia@mazums.ac.ir
(
http://dx.doi.org/10.1016/j.saa.2015.07.029
386-1425/Ó 2015 Elsevier B.V. All rights reserved.
1

A. Bekhradnia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22
19
challenges, increasing the rate of chemical reactions in organic
syntheses through microwave activation has attracted substantial
interest in recent years [16]. We wish to report microwave-
assisted procedure for the preparation of nitro-coumarin amide
derivative. Also, the effectiveness of UV–vis analysis and fluores-
cence properties of the synthesized compound and its reactivity
has been investigated toward several heavy metal cations.
(Me, t, 3H, J = 7.2), 4.5 (CH
2
, q, 2H, J = 7.2), 7.6–8.3 (m-Ar, 3H), 8.6
(@CH, s, 1H). IR (KBr): 1693 (ester C@O), 1778 (lacton C@O)
ꢂ1
1
. 3
cm . (2): m.p. 198–201 °C H NMR (400 MHz, CDCl ) d: 7.5–8.0
(m-Ar, 3H), 9.2 (@CH, s, 1H), 12.5 (OH, s, 1H). IR (KBr): 3438
ꢂ1
(OH), 1967 (acid C@O), 1776.68 (lacton C@O) cm . (3); m.p.
1
209–211 °C. H NMR (400 MHz, CDCl
3
) d: 2.5 (N(Me)
, m, 2H, JCH2-CH2 = 7.3), 2.7–2.8 (Me) NCH
CH2-CH2 = 7.3), 8.0–8.4 (m-Ar, 3H), 8.5 (@CH, s, 1H), 6.9 (NH, 1H).
2
, s, 6H),
3.7–4.0 (HNCH
J
2
2
2
, m, 2H,
IR (KBr): 3419 (amide NH), 1648 (amide NH), 1604 (amide C@O)
2
. Experimental section
ꢂ1 13
cm
.
3
C NMR (400 MHz, CDCl ) d: 35.3, 43.7, 44.7, 56.5, 114.1,
1
16.9, 117.8, 120.2, 127.7, 133.1, 138.2, 154.81, 162.3, 162.4.
2
.1. Chemicals and instruments
+
FAB-MS calcd for C14
Elemental analysis data is the following: C, 54.90%; H, 4.97%; N,
1
2
15 3 5
H N O [M+H] 306.26, found 306.00.
Melting point was measured in open capillary tubes with an
3.82%; O: 26.31 Anal. Calcd.: C, 55.08%; H, 4.92%; N, 13.77%; O:
6.23.
Electrothermal-9200 melting point apparatus. 1H and C-NMR
spectra were recorded using a Bruker (400 MHz) Avance (III) spec-
trometer. Chemical shifts (d) were reported in ppm downfield from
the internal standard tetramethylsilane (TMS). The Uv–vis spectra
were obtained using a Perkin-Elmer lambda-EZ 201 and a Jasco
FP-200 spectrofluorometer was used to record fluorescence emis-
sion spectra. Data were recorded on-line and analyzed by Excel
software on a PC computer. Fluorescence intensity measurements
were performed at room temperature. Infrared (IR) spectra in
cm
microwave irradiation reactions were carried out on a Milestone
Micro-SYNTH apparatus. Internal temperatures were measured
with fiber-optic sensor in conjunction with Milestone immersion
well.
13
3. Results and discussion
Figs. 1 and 2 show changes in the absorption and fluorescence
spectra of aqueous solution of 3, upon the addition of various
cation salts. The absorption intensity of 3 decreased with the addi-
tion of various heavy metal ions, while addition of Hg2+ produced a
higher hypochromic effect at k = 335 nm than with the other ions.
All the cations studied led to hypochromic effects in absorbance
in the UV–vis spectra of 3 (Fig. 1). By UV–vis analysis, it was diffi-
cult to identify specific cations over other metal ions because of the
ambiguous absorption spectra [17]. Therefore, to investigate the
ꢂ1
were recorded on FT-IR Perkin-Elmer spectrometer. All
2
+
selectivity of 3 for Cu in the presence of other metal ions, the flu-
orescence response 3 toward various metal ion solutions (0.3 mM)
was studied.
2.2. Synthesis of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-
chromene-3-carboxamide(3)
To obtain reliable results, the excitation spectra for compounds
6
-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carbo
In the present study, we prepared coumarin ester derivative
through the condensation of nitro-2-hydroxy benzaldehyde and
malonic ester under solvent-free conditions and microwave irradi-
ation. The produced ester was hydrolyzed to afford the related car-
boxylic acid. Subsequently, 3-carboxamide coumarin was
xamide, 3, and 3-Cu(II) were obtained and given in Fig. 3. The exci-
tation spectrum would represent the relative emission of the 3 and
3-Cu(II) in aqueous solution (HEPES:DMSO) 9:1, v/v). Since the
excitation spectrum of a fluorophore could be superimposable on
its absorption spectrum, the excitation spectra of 3 and 3-Cu(II)
were recorded at 320 nm with an emission at 420 nm.
0
produced through reaction with N,N -dimethylethylenediamine,
and its fluorescence spectra was studied in the presence of trace
+
+
+
2+
+
2+
2+
Fig. 2 shows the fluorescence spectra of 3, before and after the
amounts of cations, including Na , K , Li , Ca , Ag , Cu , Pb
,
2
+
2
+
2+
3+
2+
2+
2+
2+
2+
addition of Cu to a mixture of various cations, with excitation
at 320 nm. The fluorescence intensity of 3 increased when Cu2+
was added, with the highest fluorescence intensity observed when
Hg , Co , Cr , Mn , Fe , Ni , Cd , and Zn
.
Compound 1 was prepared by the reaction of salicylaldehyde
and diethyl malonate in the presence of piperidine and glacial
acetic acid under microwave irradiation for the time indicated in
Tables 1 (Scheme 1). Compound 2 was prepared by heating 1 in
Cu(NO
3
) solution was added (Figs. 2 and 4). In summary, the fluo-
rescence intensity was enhanced with excitation at 320 nm for
2
+
Cu over the other metal cations when 3 was used as a probe.
To investigate the binding modes between sensor 3 and its
respective cation, we carried out ab initio calculations using
HF/6-31G [18]. The optimized structure of the 3 complex is shown
in Fig. 5, indicating 1:1 stoichiometry between the sensor and Cu
In this structure, Cu binds with an oxygen atom of a carbonyl
group, a nitrogen atom of an amide, a nitrogen atom of an amine,
and two oxygen atoms of a nitrate.
alcohol-based solution. Subsequently, dicyclohexylcarbodiimide
0
(
DCC), N,N -dimethylethylenediamine, and a catalytic amount of
4
-(dimethylamino) pyridine (DMAP) were added to a stirred sus-
⁄
0
pension of 2 in chloroform. The white N,N -dicyclohexylurea
2+
.
(
DCU) precipitate was then filtered from the yellow solution, and
2
+
finally, the respective carboxamide 3 was obtained after solvent
removal and further purification.
This fluorophore was solved in (HEPES:DMSO) 9:1, v/v and eval-
uated the cation chelating by fluorescence spectroscopy. The
To find the mole percent of the sensor to moles of the metal,
UV/vis titration experiments were performed using 0.6 lmol
(2 mL 0.3 mM solution) of 3 in solution (HEPES:DMSO) 9:1 with
selected physical and spectral data for synthetic compounds are
1
as follows: (1): mp: 180–182 °C. H NMR (400 MHz, CDCl
3
) d: 1.4
ꢂ6
varying concentrations of metal nitrate salts (0–4.5 ꢁ 10 mol).
Fig. 6 shows the relationship between absorbance and a mole ratio
of 1:1, consistent with the theoretical results.
Table 1
The association constant (Ka) of compound 3 with Cu2+ was
determined using the Benesi–Hildebrand equation [19] as follows:
Microwave settings for 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-
carboxamide (1).
Step
Time
Temperature (T
1
)
Temperature (T
1
)
Max power
1
1
1
1
2
3
4
14 min
15 min
5 min
Ramp to 105 °C
105 °C
Ramp to 130 °C
130 °C
85
85
100
100
800 W
650 W
650 W
650 W
¼
Þ½Cu þꢃ þ ðFmaxꢂ
2
F ꢂ Fmin
a
K ðF
maxꢂFmin
F
min
Þ
F and Fmin represent the fluorescent intensity of the ligand 3 at mod-
erate concentration and absence of Cu , respectively. Fmax is the
5 min
+
2

2
0
A. Bekhradnia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22
O
O
O
O
OC H5
Piperidine,
AcOH
NO2
2
NO
2
H
OC H
2
5
+
Microwave(600W-850W)
OH
OC
2
H
5
O
O
1
NaOH,
EtOH,
HCl (pH=2)
O
O
O
DCC ,
NO2
DMAP
NO2
N
OH
H
N,N-dimethyl ethylene diamine
O
N(Me)2
O
O
3
2
O
O
O
NO2
Cu2+
NO2
N
H
N
O
N(Me)2
(HEPES:DMSO )
N(Me)2
O
O
Cu2+
Fluorescence
ON
Fluorescence
OFF
Scheme 1. Synthetic pathways to 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide (3).
Fig. 1. Absorption spectra of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-chromene-3-carboxamide before and after addition of heavy metal ions.
saturated fluorescent intensity of compound 3 in the presence of
Moreover, the detection limit was calculated based on the fluo-
rescence titration [20]. To determine the S/N ratio, the emission
intensity of the complex (3-Cu ) was measured and the standard
2
+
excess amount of Cu
.
2
+
According to Benesi–Hildebrand equation, the association con-
stant between 3 and Cu2 was calculated from the fluorescence
+
deviation of blank measurements was calculated. The detection
4
ꢂ1.
titration result and was found to be 3.01 ꢁ 10 M
limit was then calculated according to 3 ꢁ d /m. where db was
b

A. Bekhradnia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22
21
Fig. 4. Flourescence spectra of
3 solutions (0.3 mM) with addition of metal
solutions in aqueous solution (HEPES:DMSO) 9:1, v/v) (0.1 mM) with an excitation
at 320 nm, excitation and emission slit widths at 3.0 nm, and room temperature.
Fig. 2. Fluorescence spectra of 6-nitro-N-[2-(dimethylamino)ethyl]-2-oxo-2H-
2
+
chromene-3-carboxamide upon the addition of Cu in the presence of various
mixture of cations with an excitation at 320 nm, excitation and emission slit widths
at 3.0 nm, and room temperature.
⁄
Fig. 5. Optimized structure of 3-Cu(II) by ab initio calculation (HF/6-31G ). All
hydrogen atoms are omitted for clarity.
Fig. 6. Molar ratio of complex 3-Cu(II) by using UV Spectrum with addition of
ꢂ6
varying concentrations of the metal nitrate salts (0–4.5 ꢁ 10 mole) to 2 ml of
Fig. 3. Excitation spectra of 3 and complex 3-Cu(II) (1:1) in aqueous solution
0
.3 mM solution of 3 (0.6 lmoles) in solution (HEPES:DMSO) 9:1.
(
HEPES:DMSO) 9:1, v/v) with an emission at 420 nm, excitation and emission slit
widths at 3.0 nm, and room temperature.
4
. Fluorescence and absorption spectra
the standard deviation of blank solutions, and m was the slope
between intensity vs. sample concentration. The detection limit
Stock solutions of 3 (0.3 mM) was prepared in aqueous solution
(HEPES:DMSO) 9:1 v/v. Stock solutions of metal nitrate salts
2
+
ꢂ6
for Cu was calculated to 2.7 ꢁ 10 mol/L. For the biological
application of the ligand, the sensing should perform in physiolog-
ical pH (6.8–8.2). Therefore, all of experiments were done in the
aqueous solution (HEPES:DMSO) 9:1, v/v).
+
+
+
2+
+
2+
2+
2+
2+
3+
2+
(0.1 mM Na , K , Li , Ca , Ag , Cu , Pb , Hg , Co , Cr , Mn
,
2
+
2+
2+
2+
Fe , Ni , Cd , or Zn ) was prepared in water. The prepared metal
ꢂ6
solutions (4.5 ꢁ 10 mol or 45 mL 0.1 mM solution) were mixed

2
2
A. Bekhradnia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 18–22
ꢂ7
with solution of 3 (6.0 ꢁ 10 mol or 2 mL 0.3 mM solution), and
the absorption and fluorescence intensities of the free ligand and
complexed form of the probe compound were measured. The
UV–vis absorbance and fluorescence spectra of receptor varied
with the concentration of metal ions. Fluorescence quantum yield
was determined by comparing the emission and absorption inten-
sities of the probe with those of a fluorescence standard, fluores-
cein in 0.1 N NaOH. For all fluorescence measurements, the
excitation wavelength was 320 nm, while for excitation spectra,
the emission wavelength was 400 nm, with excitation and emis-
sions slit widths of 3.0 nm. Quantum yield of 12.1% was calculated
for the 3-Cu(II) complex. Nevertheless, low quantum yields were
obtained for combined receptor and other cations as well as free
receptor and no worth to report.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2015.07.029.
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