Synthesis and characterization of p-dimethylaminobenzaldehyde benzoylthiourea and study towards selective and sensitive fluorescent sensor for detection of iron (III) cation in aqueous solution

Benzoyl thiourea derivatives ligand backbones contain Oxygen (O), Nitrogen (N), and Suphur (S) donor atoms can react with transition metal ions and form stable metal complexes. A new ligand was synthesized by the reaction of benzoyl isothiocyanate with diethylenetriamine and characterized by using Elemental Analysis (EA), Infrared (IR) spectroscopy and proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. The monofunctionalize ligand were synthesized using 1:1 condensation of p-dimethylaminobenzaldehyde with diethylenetriamine and confirmed by disappearance of carbonyl group, C=O at 1656 cm-1 with the appearance of peak C=N in the range 1633 - 1638 cm-1 in the IR spectrum. This monofunctionalize ligand was synthesis by using Schiff base technique. Benzoyl thiourea derivatives, HN are synthesis from the 1:1 condensation of benzoyl isothiocyanates with monofunctionalized ligand. HN ligand was verified by the presence of peaks v(N-H), v(C=O), v(C=N) v(C-N) and v(C=S) at 3317 - 3336 cm-1, 1612 -1660 cm-1, 1550 -1589 cm-1, 1234-1366 cm-1 and 709-767 cm-1 respectively while 1H NMR show peaks of alkane (CH2), benzene (Ar-H), CONH, CSNH at 3.75, 6.73 – 7.33, 8.19, and 8.25 respectively. The total percent composition of C, H, N, O, and S using Elemental Analysis for HN, C21H27N5OS found was 85.23%. For application, further study on selectivity and sensitivity of HN ligand was conducted on several metal ions. The fluorescent emission spectroscopy shown that HN is a selective fluorescent sensor for Fe3+ and Cu2+ ions but not for other metal ions such as Co2+, Ni2+, Mn2+, Cr3+ and Zn2+. However, HN ligand shows more sensitive towards Fe3+ ions than Cu2+ ions which provide rapid detection of Fe3+ ions at concentrations as low as 4.5 x 10-7 M than 2.9 x 10-6 M of Cu2+ ions. As conclusion, HN ligand was expected to be useful as efficient chemical sensor for detection of Fe3+ ion.


Introduction
Nowadays, the development of chemosensors for selectivity and sensitivity quantification of environmentally and biologically for heavy and transition metal ions, has attracted a high attention [1]. They usually represent an environmental concern when present in uncontrolled amounts, but at the same time some of them such as iron, zinc, manganese, copper and cobalt are present as essential elements in biological systems. Among biologically important metal ions, iron is the most abundant transition metal ion present in the human body. As one of the most essential trace elements in biological systems, Fe 3+ performs a major role in many biochemical processes. Fe 3+ ion is an important and essential for proper functioning of all living cells and acts as a cofactor in many enzymatic reactions to human as well as in specialized transport and storage of proteins. The deficiency of Fe 3+ causes anaemia, liver damage, diabetes, hemochromatosis, Parkinson's disease and cancer [2]. However, iron overload is a clinical condition in which there is an excess of iron levels in the organism typically due to abnormally high iron absorption from the diet, periodical blood transfusions or wrong use of iron by the organism as result of the blocking action of determined cytokines [3]. If continued in time, high levels of Fe 3+ within the body have been associated with increasing incidence of certain cancers and dysfunction of certain organs, such as heart, pancreas and liver. In recent years, several fluorescent sensors have been developed for the detection of transition and heavy metal ions, such as dimethylaminocinnamaldehyde-aminothiourea (DA) for detecting Ag + , Hg 2+ and Cu 2+ [4], pdimethylaminobenzaldehyde thiosemicarbazone for detecting Hg 2+ [5], rhodamine derivatives (RBS) for detecting Cu 2+ and F - [6] and also salicylaldimine based receptor sensing of multi metal ions and anions [7]. Meanwhile, various sensors for Fe 3+ have been reported [8][9] [10]. However, to the best of our knowledge, the static quenching mechanism for aminothiourea-based chemosensor to monitor trace levels of iron ions is rarely reported. Fluorescence-based analytical methods are widely used for detection of metal ions in an early stage because they are sensitive, simple and less expensive. In this study, the fluorescent sensor for sensitive determination of iron is reported. In design of fluorescent sensors for these ions, aminothiourea is a preferred receptor because of its strong affinity [4]. Scheme 1 below shows the reaction of synthesis of ligand HN step by step. Step

Apparatus and reagents
The inorganic salts Fe(Cl2)3.6H2O, Mn(SO4)2, Cr(SO4)26H2O, Co(Cl2)2.6H2O, Ni(NO3)2.6H2O, Cu(NO3)2.3H2O and Zn(Cl2)2 were purchased from Sigma Aldrich. All solvents and other reagents were of analytical grade. 1 H NMR spectra are recorded on a Bruker Avance III 300 Spectrometer at room temperature. 1 H chemical shifts are reported in ppm downfield from tetramethylsilane (TMS), d scale with the solvent resonances as internal standards). Melting points were measured using BÜCHI Melting Point B-545 and the elemental analyses were conducted using CHNS Analyzer Flash EA 1112 series. The infrared spectra were performed using FTIR Perkin Elmer 100 Spectrophotometer in the spectral range of 4500-600 cm -1 .

General Procedure for Fluorescence spectrometer
The fluorescence spectra were recorded on Luminescence Spectrometer LS 55 equipped with quartz cuvettes of 1 cm path length. The excitation and emission slit widths were 10 nm. Stock solutions for analysis were prepared (0.1 M for Ligand HN (Acetonitrile/H2O, 9:1 (v/v), pH = 7.0) immediately before the experiments. The fluorescence titration was performed with a series of 5 × 10 -5 M solutions of HN containing various equivalents of Fe 3+ ions. Binding studies were confirmed by job plot. The association constant (Ka) were calculated by the linear Benesi-Hildebrand equation from fluorescence (Eq. (1)) study by, Where F, F₀ and Fmin are the emission intensities, respectively in the absence and infinite concentration of the metal ion (Fe 3+ ).

Sensing in the presence of competing ions.
Ligand HN was applied as chemosensor by using Fluorescence Emission Spectroscopy (FES).to observe several parameters such as selectivity, sensitivity, stoichiometry and binding mode studies and also the competition. For an excellent chemosensor, high selectivity is a matter of necessity. Among the various metal ions, the fluorescence emission spectra showed a noteworthy high selectivity to Fe 3+ and Cu 2+ with respect to the fluorescence quenching. Other metal ions developed no distinct significant fluorescence intensity changes. Related metal ions, including Fe 3+ , Cu 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cr 3+ and Zn 2+ , were used to evaluate the metal ion binding properties of HN in ACN/ H2O (10:1/ v:v, pH 7.0) solution by fluorescence spectroscopy (Fig. 1). HN can detect both Fe 3+ ion and Cu 2+ ion but HN more sensitive sensor towards Fe 3+ ion than Cu 2+ ion. Figure 2 shows the results of fluorescence titration of HN upon the gradual addition of Fe 3+ (Fig.  2.(a)) and Cu 2+ (Fig 2.(b)) in ACN/H2O solution under excitation at λ = 561 nm. A linear relationship was observed to exist between the relative fluorescent intensity of HN and the concentration of Fe 3+ in the range of 5 x 10 -7 M-17.5 x 10 -7 M with a detection limit of 4.5 x 10 -7 M while the concentration of Cu 2+ in the range of 1 x 10 -5 M -4 x 10 -5 M with a detection limit of 2.9 x 10 -6 M. According to the U.S. Environmental Protection Agency (EPA), the maximum content of iron in drinking water is ~5.357 µM. Therefore, detection of trace irons becomes very important in the chemical research [11].

Competition of Fe 3+ toward other metal ions
The competition experiment, which was carried out by adding Fe 3+ to HN solution in the presence of other metal ions (Fig. 4), showed that the Fe 3+ -induced fluorescence response was not interfered by commonly coexistence ions. The result suggested that probe HN showed a remarkable selectivity toward Fe 3+ over other competitive ions. However, Cu 2+ also showed a remarkable selectivity but not as sensitive as Fe 3+ after we used all the metal ions in 5 x 10 -6 M.

Conclusion
In conclusion, HN bearing thiourea moiety as the binding site and p-dimethylaminobenzaldehyde moiety as the signal group was designed and synthesized to apply as fluorescent sensor for Fe 3+ ion. This sensor showed specific selectivity for Fe 3+ in ACN/H2O binary solutions indicated that the aminobenzaldehyde moiety acted as a signal group (fluorophore) and played a crucial role in the process of fluorescent recognition. Investigation of the binding constant mechanism indicated that the sensor HN recognized Fe 3+ by forming a stable 1:1 HN-Fe 3+ complex. The coexistence of other cations did not interfere with the Fe 3+ recognition process unless for Cu 2+ . HN also sense the Cu 2+ but not as sensitive as Fe 3+ . Moreover, the detection limit of the sensor HN toward Fe 3+ was 4.5 x 10 -7 M and 2.9 x 10 -6 M toward Cu 2+ . This indicated that the sensor HN may be useful for preliminary detection of Fe 3+ ion by simple solvent extraction in chemical and environmental applications.