A sensitive fluorescent chemosensor was developed for detecting CuII through microwave irradiation by using diethylamino-3-carboxamide coumarin. This receptor was employed as fluorescent probe for CuII with selectivity over other cations in aqueous solution. The fluorescence intensity quenched when 7-diethylamino-N-[2-(dimethylamino) ethyl]-2-oxo-2H-chromene-3-carboxamide (3) used as a receptor in the presence of very low concentration of CuII with an excitation at 360 nm.
Keywords: Fluorescent chemosensor; Cation detection; Coumarinbased fluorescent probe
Coumarins (benzopyran-2-ones) as oxygenated heterocycles with natural base possess several biological activity consist of anticancer, antioxidant, anti-inflammatory, MAO inhibitory, antiviral, antimicrobial and antihyperlipidemic activity . Several methods for synthesis of coumarins have been developed in recent years. Some of the most common synthetic reactions for oxygenated heterocycles are Pechmann and Duisberg , Perkin and Henry , Brafola et al. , Claisen et al. , Shringer , Heck, and 1,3-dipolar cycloaddition reactions .
The copper plays an important role in various biological processes . However, copper is released into the bloodstream and is deposited in the kidneys, cornea, and particularly, the brain [11,12]. Therefore, exposure to higher levels of copper can cause rheumatoid arthritis, gastrointestinal disturbances, and Wilson’s disease.
Copper ion has an essential role in various enzymes involve cytochrome oxidase, superoxide dismutase, dopamine-hydroxylase and catalysis . Moreover, anticancer activity and DNA-binding  of the CuII has been investigated by coumarin derivatives .
Despite these functional roles, over load of this metal have toxic challenges in human health which have been visible in Huntington’s disease , Alzheimer ’s disease (AD)  and Parkinson’s disease (PD) .
The average diet provides substantial amounts of copper, and the recommended intake is 0.9 mg/day. Also, the average concentration of blood copper in the normal people is 100-150 μg/dL. Thus, detection and elimination of this ion is important. In this regard, many analytical techniques are available for the determination of species. Among them, substantial attention has been paid to fluorometry technique because of its high sensitivity [19-22].
Therefore, we designed a novel derived coumarin fluorescent sensor for selective detection of CuII through solvent-free condition.
1H and 13C-NMR spectra were recorded using a Bruker (400 MHz) Avance (III) spectrometer. Chemical shifts (δ) were reported in ppm downfield from the internal standard tetramethylsilane (TMS). The fluorescence emission spectra were obtained using a Jasco FP-200 spectrofluorometer. The Perkin-Elmer lambda-EZ 201 was used to record UV–Vis spectra.
The FT-IR Perkin-Elmer spectrometer was employed to record Infrared (IR) spectra in cm-1. 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. Electrothermal-9200 melting point apparatus was used to measure melting point in open capillary tubes.
1-1-7-diethyl)-N-(2-(dimethylamino)ethyl)-2-oxo-2H-chromene- 3-carboxamide Coumarin ester derivative was prepared through the condensation of 4-(Diethylamino)salicylaldehyde and malonic ester under microwave irradiation. The prepared ester was hydrolyzed to produce the related carboxylic acid. Consequently, 3-carboxamide coumarin, as a receptor, was easily made through reaction with N,N‘- dimethylethylenediamine. Fluorescence spectra was investigated in the presence of low concentration of cations, including NaІ, KІ, CuІІ, PbІІ, HgІІ, CoІІ, ZnІІ, FeІІ, CdІІ, AlІІІ and CrІІІ. Compound 1 was formed by the reaction of 4-(Diethylamino)salicylaldehyde and diethyl malonate in the presence of piperidine and glacial acetic acid under solventfree conditions (Table 1, Scheme 1). The related carboxylic acid 2 was produced by hydrolysis of 1 in sodium hydroxide solution.
Scheme 1: Synthetic pathways to 7-diethylamino-N-[2-(dimethylamino)ethyl]- 2-oxo-2H-chromene-3-carboxamide .
|Step||Time||Temperature T1)||Temperature T2)||Max Power|
|1||15 min||Ramp to 110°C||85||800 W|
|2||17 min||110°C||85||700 W|
|3||7 min||Ramp to 135°C||105||700 W|
|4||6 min||135°C||105||700 W|
Table 1: Microwave settings for compound .
In order to produce the desired carboxamide 3, dicyclohexylcarbodiimide (DCC), N,N‘-dimethylethylenediamine, and 4-(dimethylamino) pyridine (DMAP) as a catalyst were added to a mixture of 2 dissolved in chloroform. After filtration of N,N‘- dicyclohexylurea (DCU) precipitate, the carboxamide 3 was obtained after purification in mixture of ethanol and water. Subsequently, spectrofluorometry was used to assessment of the cations chelating on receptor 3 in (HEPES:DMSO) 9:1, v/v.
The selected physical and spectral data for synthetic compounds are as follows: (1): mp: 195–199ºC. 1H NMR (400 MHz, CDCl3) δ: 1.1(Me, t, 6H, J=7.2), 1.5(Me, t, 3H, J=7.0), 3.2 ( CH2, 4H, q, J=7.2), 4.3 (CH2, q, 2H, J=7.0), 6.0 (Ar, d, 1H), 6.1 (Ar, dd, 1H), 7.1 ( Ar, d, 1H), 9.3 (Ar, s, 1H). IR (KBr): 1685 (ester C=O), 1769 (lacton C=O) cm-1. (2): m.p. 201–205ºC. 1H NMR (400 MHz, CDCl3) δ: 1.1(Me, t, 6H, J=7.2), 3.2 (CH2, 4H, q, J=7.2), 6.1 ( Ar, d, 1H), 6.3(Ar, dd, 1H), 7.3 ( Ar, d, 1H), 9.3 (Ar, s, 1H), 11.3 (OH, s, 1H). IR (KBr): 3445 (OH), 1960 (acid C=O), 1786 (lacton C=O) cm-1. (3); m.p. 212–215ºC. 1H NMR (400 MHz, CDCl3) δ: 1.1(Me, t, 6H, J=7.2), 2.4 (N(Me)2, s, 6H), 3.2 (CH2, 4H, q, J=7.2), 3.8–4.1 (HNCH2, m, 2H, JCH2-CH2=7.1), 6.1 ( Ar, d, 1H), 6.3 (Ar, dd, 1H), 6.8 (NH, 1H), 7.3 (Ar, d, 1H), 9.3 (Ar, s, 1H).
IR (KBr): 3425 (amide NH), 1652 (amide NH), 1610 (amide C=O) cm-1. 13C NMR (400 MHz, CDCl3) δ: 12.1, 37.1, 43.2, 45.2, 46.8, 57.2, 115.1, 116.7, 118.2, 121.0, 129.2, 135.2, 140.1, 155.1, 16.1.2, 163.1.
UV–Vis spectra show hypochromic effects in absorbance for all the cations studied in this research. Nevertheless, it was difficult to detect specific cations because of the no selectivity related to any cations. Thus, spectrofluorometry was used to investigate of the CuII chelating on fluorophore 3 in the presence of other cations.
The fluorescence spectra for complex 7-diethyl)-N-(2- (dimethylamino) ethyl)-2-oxo-2H-chromene-3-carboxamide and cation, 3-Mn+, was studied and given in Figure 1 in aqueous solution (HEPES:DMSO) 9:1, v/v). The fluorescence spectra 3-Mn+ was recorded at 360 nm with an emission at 470 nm. Also, Figure 2 shows the fluorescence spectra of 7-(N,N-diethyl)-N-(2-(dimethylamino)ethyl)- 2-oxo-2H-chromene-3-carboxamide upon the addition of CuІІ in the presence of various mixture of cations with an excitation at 360 nm.
To investigate of stoichiometry between the sensor and Cu25, spectroscopic changes generated upon titration experiments were done using 0.6 μmol of 3 in solution (HEPES:DMSO) 9:1 with changing concentrations of metal salts (0-4.5 μmol) by means of UV-visible spectroscopy (Figure 3).
Furthermore, full geometry optimization were performed by means of Hartree–Fock (HF) and 6-31G* basis set employing the Gaussian 03 code [23,24]. To assess the performance of this approach, the 3-CuІІ structure was proposed and computed at abinitio method (Figure 4) .
Coumarin-based derivatives are important group of fluorescent heterocycles that can be used as fluorescent probes [26-29]. Recently, we have synthesized nitro-3-carboxamide coumarin derivative, proposed as novel fluorescent chemosensor and has been employed as fluorescent probe for CuІІ . That receptor played a role for complexation of heavy toxic metals and exhibit enhanced fluorescence in the presence of CuІІ. In this research, we prepared and studied a new fluorescent chemosensor that CuІІ over other ions was detected by florescence quenching technique. In fact, the fluorescence intensity decreased in the presence of CuІІ in comparison of other metal ions (Figure 1). Thus, fluorescence quenching was occurred when CuІІ gradually added (Figure 2).
Figure 3 shows the Uv-Vis titration of complex sensor-CuІІ using 0.6 μmol of 3 in solution (HEPES:DMSO) 9:1 with changing concentrations of CuІІ salts (0-4.5 μmol). The mild negative slope of the plotted line during the initial phase indicates a low concentration of 3 in the vessel and forming 3-CuІІcomplex. Increasing of CuІІ concentration, the slope became linear, representing the highest concentration of 3-CuІІ complex and the ligand 3 was consumed. It shows 1:1 stoichiometry between the sensor and CuІІ.
To find reliable results for investigation of binding modes between fluorophore and its CuІІ, ab initio calculations was done using HF/6- 31G*. The optimized model of 3-CuІІcomplex is revealed that CuІІ binds with an oxygen atom, two nitrogen atoms of an amide and an amine, as well as two oxygen atoms of an anion (Figure 4).
As a result, the ab initio calculation outcomes confirm the relationship between absorbance and a mole ratio of 1:1 stoichiometry.
The association constant (Ka) of complex sensor-CuІІ was found using the Benesi–Hildebrand equation  as follows:
F and Fmin represent the fluorescent intensity of the ligand 3 at moderate concentration and in the presence of excess amount of CuІІ, respectively. Fmax is the saturated fluorescent intensity of free ligand 3.
Regarding to Benesi–Hildebrand evaluation, the association constant for complex sensor-CuII was calculated from the spectrofluorometeric titration data and was found to be 7.2 × 104 M−1.
These results led us to the agreement of the Benesi-Hildebrand evaluation method and abinitio study for the determination of association constant with 1:1 stoichiometry between 3 and CuІІ.
Here, a new coumarin-based fluorescent chemosensor through microwave irradiation as fluorescent probe for CuІІ ion was designed and synthesized. Dimethylamino-N-[2-(dimethylamino)ethyl]-2-oxo- 2H-chromene-3-carboxamide (3) is a suitable fluorophore because of its large stokes shift and visible excitation and emission wavelengths. The receptor exhibits quenched fluorescence in the presence of CuІІ with selectivity over other metal ions in aqueous solution. The lowest fluorescence intensity for diethylamino-3-carboxamide coumarin was observed in the presence of CuІІ in preference to a variety of other common heavy and toxic metal ions. Therefore, we synthesized a new diethylamino-3-carboxamide coumarin as a receptor, which employed for detection of CuІІ with excitation at 360 nm.
The authors gratefully acknowledge the financial support for this work received from the Mazandaran University of Medical Sciences ‘‘Professor’s Projects Funds’’.