A novel selective fluorescent chemosensor for Fe 3+ ions based on phthalonitrile dimer: synthesis, analysis, and theoretical studies

Phenyl-4,4-di(3,6-dibutoxyphthalonitrile) ( 3 ) was synthesized by the reaction of 1,4-phenylenebisboronic acid ( 1 ) and 4-bromo-3,6-dibutoxyphthalonitrile ( 2 ), using Suzuki cross-coupling reaction. The newly synthesized compound ( 3 ) was characterized by FT-IR, MALDI-MS, ESI-MS, 1 H-NMR, 13 C-NMR, and 13 C-DEPT-135-NMR. The fluorescence property of phenyl-4,4-di(3,6-dibutoxyphthalonitrile) ( 3 ) towards various metal ions was investigated by fluorescence spectroscopy, and it was observed thatthe compound ( 3 ) displayed a significantly ‘turn-off’ response to Fe 3+ , which was referred to 1:2 complex formation between ligand ( 3 ) and Fe 3+ . The compound was also studied via density functional theory calculations revealing the interaction mechanism of the molecule with Fe 3+ ions.

In the FT-IR spectrum of the phthalonitrile compound (3), the vibration peaks were obtained between 3090 and 3055 cm -1 belonging to aromatic-CH stretching, and between 2957 and 2872 cm -1 belonging to aliphatic-CH stretching. As a result of Suzuki cross-coupling reaction, the broad peak of 1,4-phenylenebisboronic acid (1) for the -OH band disappeared, and the most characteristic peak for -C≡N stretching was observed as a sharp peak at 2227 cm −1 for the phthalonitrile compound (3).
The 1 H-NMR spectrum of the phthalonitrile compound (3) was measured in DMSO-d 6 . This spectrum exhibited characteristic signals for the aromatic protons between 7.61 and 7.51 ppm. Besides, the aliphatic protons were displayed as a triplet peak at 4.15 ppm for -O-CH 2 protons, as multiplet peaks between 1.71 and 1.44 ppm for -CH 2 protons, and as a triplet peak at 0.94 for -CH 3 protons (Figure 1a).
The 13 C-NMR spectrum of phthalonitrile compound (3) complied with the expected structure. 13 C-NMR spectrum is shown in Figure1b, which shows signals of aromatic carbons between 103.22 and 155.37 ppm, and aliphatic carbons between 14.07 and 69.97 ppm. The carbon atom of the -C≡N group on the phthalonitrile was observed at 121.06 ppm. Figure 1c shows 13 C-DEPT-135 spectrum of phthalonitrile compound (3) in DMSO-d 6, and only the proton bearing carbons are defined at this spectrum. In DEPT 135 spectrum of this compound, 13 C signals arising due to methyl (-CH 3 ) and methine (-CH) appear positive, methylene as negative (-CH 2 ) whereas no quaternary carbons show up.
The molecular ion peak of the phthalonitrile 3 was observed at 618.510 as [M] + by MALDI-TOF/TOF (Figure 2a). Two equivalents of Fe 3+ solution was added to phthalonitrile compound (3), which dissolved in acetone, for detecting metal-ligand complexes by using ESI-MS. The molecular ion peak of phthalonitrile compound (3) (Figure 2b).

Chemosensor properties of phthalonitrile 3 to metal ions
The absorption and fluorescence properties of phthalonitrile compound (3) were studied in DMSO by UV-Vis absorption and fluorescence spectrophotometers, respectively. Broad absorption bands were observed between the ranges of 330-350 nm in the UV-Vis spectrum of this phthalonitrile compound ( Figure 3). The fluorescence intensity maximum of phthalonitrile compound (3) was observed at 397 nm when excited at 340 nm in DMSO solution ( Figure 3).
The effect of time on fluorescence signal change of phthalonitrile compound (3) for the detection of Fe 3+ was evaluated between 2-40 s (Figure 6a). The relative fluorescence intensity of phthalonitrile compound (3)-Fe 3+ complex was not found stable until the 20th s, and then it was nearly unchanged after this time. Therefore, this time is sufficient as the detection time of Fe 3+ for consistent and accurate results. The photostability of phthalonitrile compound (3) and its Fe 3+ complex was evaluated between 0 and 60 min at daylight (Figure 6b). Fluorescence signals of the phthalonitrile compound (3) and its Fe 3+ complex did not change and remained stable until the 60th min. The reversibility of the detection process of Fe 3+ with    the phthalonitrile compound (3) was evaluated with EDTA and ascorbic acid after the complexation process (Figure 6c) [22]. The quenched fluorescence signal of the phthalonitrile compound (3) after the addition of Fe 3+ was not restored by the addition of EDTA and ascorbic acid, which showed that the Fe 3+ recognition is an irreversible process [23].
Fluorescence titration of the phthalonitrile compound (3) with Fe 3+ was performed to understand the binding mode of phthalonitrile compound (3) with Fe 3+ (Figure 7). For that purpose, a gradually increased amount of Fe 3+ up to 500 μM was added to DMSO solution of phthalonitrile compound (3). As can be seen in Figure 7, fluorescence signal of the phthalonitrile compound (3) which was observed at 397 nm, was proportionally decreased by the increased concentration of Fe 3+ , and finally it was completely quenched by the addition of 500 μM Fe 3+ . Linear response change of the phthalonitrile compound's (3) fluorescence intensity by the addition of Fe 3+ at 397 nm is shown in Figure 7 inset, which was used for the calibration curve for Fe 3+ analysis. The linear regression equation was found as follows: F= -1.6967[Fe 3+ ] + 805.46 for 2-500 μM ofFe 3+ (R 2 = 0.9938). The limit of detection (LOD) and the limit of quantification (LOQ) were calculated according to 3σ/k and 9σ/k, respectively. These values were found as 3.28 μM and 9.85 μM, respectively, which pointed out high sensitivity [24,25]. Precision is an important parameter for fluorescence sensor applications. Therefore, the precision of the phthalonitrile compound (3) was determined using ten measurements for 500 μM Fe 3+ under optimum conditions, and the relative standard deviation (RSD%) was calculated as 2.81% for Fe 3+ .
The formation of the complex between phthalonitrile compound (3) (C = 2 µM) and Fe 3+ ion was experimentally indicated by performing a Job's plot analysis. The titration of the phthalonitrile compound (3) with Fe 3+ cation showed a decrease in the fluorescence intensities by the increasing concentrations of Fe 3+ cation (C = 2 µM). In good agreement with theoretical calculations, the Job's plot shows a maximum at 0.6 indication of a 1:2 stoichiometry between phthalonitrile 3 and Fe 3+ ion in DMSO ( Figure 8). As it is expected, considering the Job's plots and association constants in accordance with 1:2 stoichiometry, the possible binding mode between phthalonitrile compound (3) and Fe 3+ is proposed in Figure 9. The strong fluorescence intensity of this phthalonitrile 3 was turn-off after the addition of Fe 3+ ions. After the investigation of binding properties of phthalonitrile compound (3) by the addition of Fe 3+ , the practical determination of Fe 3+ in industrial wastewater was carried out using spike/recovery test, and the results were calculated via calibration curve. As shown in Table 1, phthalonitrile compound (3) was found able to determine different concentrations  of spiked Fe 3+ with good recovery, indicating that phthalonitrile compound (3) can potentially be employed for detecting Fe 3+ in real samples. In addition, analytical parameters of phthalonitrile compound (3) for Fe 3+ determination were compared with some other Fe 3+ -selective fluorescent sensor studies [26][27][28][29][30]. As seen in Table 2, the phthalonitrile compound (3) showed lower LOD and larger linear range than the given fluorescent sensors studied in the literature. This comparison demonstrated that the presented phthalonitrile compound (3) is a selective, sensitive, and an accessible alternative fluorescent sensor for the detection of Fe 3+ ions.

Computational studies
The interaction mechanism of Fe 3+ atom with phthalonitrile compound (3) was investigated via density functional theory. After optimizing the structure of the molecule, various possible interaction sites for Fe 3+ ion are scanned by placing a single Fe 3+ ion and letting the geometry optimize without any constraints. Figure 10a shows various possible interaction sites after the geometry optimization process. The adsorption energy for various sites are calculated using the formula E ads = E(molecule + ion) -E(ion) -E(molecule), where E(ion) and E(molecule) are the total energies of isolated ion and molecule, respectively, while E(molecule+ion) represents the total energy of the optimized molecule + ion system. Table 3 shows the interaction energy values for different sites on the molecule. While energetically the most stable interaction site is found to be between the two cyanide groups, this site corresponds to a small fraction of the total surface area of the molecule. It is also worth to note that the interaction energy of site 4 is exactly twice as the energy of site 3, where Fe 3+ ion interacts with a single cyanide group, which indicates that the highest interaction energy of site 4 results from the duplication of a CN-Fe 3+ interaction, which is not as high as the hexagon-Fe 3+ interaction by itself. Moreover, the hexagons of the molecule constitute a significant fraction of the total surface area of the molecule, so it can be deduced that the most active sites of phthalonitrile molecule (3) are the ones around carbon rings. It is also clear that the Fe 3+ ion refuses to bind in sites 5 and 6, which are totally saturated carbon chains. Looking at the geometry, one can deduce that the interaction has a more physisorption character rather than a chemical activation.  We also investigated the electronic structure of the pure phthalonitrile molecule in the context of the frontier molecular orbital theory. A typical isosurface of HOMO state wavefunction is shown in Figure 10b, which is seen to be distributed over the active sites-especially carbon rings. This verifies our energetics analysis above.

Conclusion
In summary, the novel phenyl-4,4-di(3,6-dibutoxyphthalonitrile) (3) was designed and synthesized as a new fluorescence chemosensor for Fe 3+ . This phthalonitrile compound (3) was characterized by different spectroscopic methods, such as FT-IR, MALDI-MS, ESI-MS, 1 H NMR, 13 C NMR, and 13 C-DEPT-135 NMR. In addition to these, the impacts of metal ions on the fluorescence attitude of the studied compound (3) were investigated to see whether this compound can be used as a chemosensors for metal ions. An important decrease in the fluorescence emission by the addition of the Fe 3+ cation was observed. Also, since change of color is observed in the solution when Fe 3+ was added, the perfect fluorescent response to Fe 3+ in DMSO can be detected even by the naked eye. Thanks to these results that were obtained, it can be asserted that this compound (3) designed as a metal-ion sensor has the potential to be used for a variety of chemical and biological applications in the future. Table 3. Interaction energies of Fe 3+ ion on phthalonitrile molecule for different interaction sites defined in Figure  10a.

Interaction site
Interaction energy (eV)