An anionic chromogenic chemosensor based on 4–(4–nitrobenzylideneamine)–2,6–diphenylphenol for selective detection of cyanide in acetonitrile–water mixtures

4–(4–Nitrobenzylideneamine)–2,6–diphenylphenol 3a was synthesized and studied as an anionic chromogenic chemosensor. Solutions of 3a in acetonitrile are colorless but turn blue under deprotonation. From the various anions added to the solutions of 3a only CN – , F – , and with less intensity CH 3 COO – and H 2 PO 4– , led to colored solutions. With the addition of up to 2.4% (v/v) of water, of all the anions used only CN – was able to act as a base and cause a change in the color of the solution. A model was used to explain the results, based on two 3a :anion stoichiometries, 1:1 and 1:2.


Introduction
The recognition and detection of anions is a field which has attracted increasing interest in recent years due to the fundamental importance of these species in many chemical and biological processes. 1Therefore, many methodologies based on optical chemosensors have been designed to perform selective naked-eye and quantitative detection of anionic species. 1 A very simple strategy used for the development of anionic chemosensors involves the study of molecules whose color in solution changes with the addition of a particular anion to the medium.The color change observed is due to an alteration in the molecular structure of the chemosensor, its selectivity toward an anionic species being related to the ability of the anions to act as bases, which defines a differentiated level of interaction with the receptor site in the chemosensor, through hydrogen bonding (HB) or by means of proton transfer processes.This strategy has been used in the recent years to study chromogenic chemosensors for more basic anions, such as F -and CN -, in non-hydrogen bonding donor (non-HBD) solvents, being of particular importance in the case of F -, 2 which exhibits a greater effect compared with other anionic species due to its smaller size, higher charge density, and higher electron affinity.This makes the anion capable of forming strong interactions with, for instance, the hydroxylic group in phenols.The development of simple F -chemosensors is of great interest considering the importance of this anion in the clinical treatment of osteoporosis, the role it plays as an environmental pollutant and the diseases related to its over-accumulation in bones. 3nother interesting anion in terms of detection is CN -, which is lethal in very small amounts due to its ability to bind strongly to the active site of cytochromo-oxidase, leading to the inhibition of the mitochondrial electron transport chain, and to a decrease in the oxidative metabolism. 4CN -has many applications in metallurgy, fishing, mining, and the fabrication of polymers.Some fruit seeds and roots release CN -through hydrolysis. 4In addition, many chemical warfare compounds, such as sarin, soman, and tabun, 5 deliver F -and CN -through hydrolysis and this is important in relation to developing chemosensors for the detection of these neurotoxic agents. 6][9][10][11] The connection of the phenol donor group to an acceptor group by means of a conjugated bridge creates an interesting feature since the deprotonation of the compound generates a colored conjugated base.The acidity of the chemosensor can be, in principle, modulated through the effect of the medium polarity and modifications in the molecular structure of the compound. 10,11Hong and co-workers used azophenols as F --selective chromogenic chemosensors, studying the ability of the anions to interact with the chemosensor in trichloromethane through HB according to their basicity. 7e have previously studied chemosensors with solvatochromic merocyanines, such as Reichardt's betaine, 2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1-yl)phenolate 1, and 4-[(1methyl-4(1H)-pyridinylidene)-ethylidene]-2,5-cyclohexadien-1-one, known as Brooker's merocyanine 2, as signalizing units.Many studies have been carried out with these dyes due to the fact that they are solvatochromic, i.e., their UV-vis spectrum changes when the medium polarity is altered. 12,13Besides the classical applications of these dyes, they have been recently used as signalizing units in the development of chemosensors for anionic [9][10][11]14 and neutral 15 analytes. In his regard, we have shown that 1, in chloroform, in its protonated form, acts as an anionic chemosensor for F -and H2PO4 -. 9 These anions are able to abstract a proton from the phenol group of the protonated dye, leading to colored solutions.Protonated 1 was also able to signal the presence of I -through the complexation of the anion with the pyridinium group. 9 study was carried out in trichloromethane to find a protonated pyridiniophenolate able to operate in a biphasic system, aiming at the selective detection of CN -in aqueous solution.11 Compounds 1 and 2, both in their protonated form, were studied in acetonitrile and acetonitrile-water mixtures as anionic chemosensors.10 Protonated 1 has potential for the recognition and visual detection of CN -, F -, and H2PO4 -in acetonitrile.10 The titration of the dye in its protonated form with the anions revealed two chemosensor:anion stoichiometries, one 1:1 and the other of the uncommon 1:3 type.The data suggested that the anion would be capable of forming firstly an ion pair with the pyridinium center in the chemosensor and after that a second equivalent of the anion would be needed to form a complex with the phenolic proton through HB.Finally, a third equivalent of the anion would be needed for the abstraction of the proton, with the formation of an [HA2] -complex.10 With the addition of small amounts of water to the system, selectivity for CN -was observed in relation to the other anions. Aso, the addition of water to the system leads to a decrease in the magnitude of the binding constants related to the 1:3 stoichiometry, due to the action of water, which preferentially solvates the anion and inhibits its interaction with the pyridinium center.10 In order to obtain further experimental results to corroborate the proposal presented for the 1:3 chemosensor:anion stoichiometry observed for systems with 1 and 2, as well as to obtain more efficient novel chromogenic systems with more simple molecular structures than 1, this paper investigates the use of compound 3 as an anionic chemosensor.Compound 3, 4-(4nitrobenzylideneamine)-2,6-diphenylphenol, has a different acceptor group from that observed for compounds 1 and 2 and this should reflect in a change in the pattern of the chemosensor:anion stoichiometries, without altering in the efficiency of its anion sensing properties.

Results and Discussion
Figure 1 shows the influence of various anions (HSO4 -, H2PO4 -, NO3 -, CN -, CH3COO -, F -, Cl -, Br -, and I -) on the color of the solutions of 3a in acetonitrile and acetonitrile-water mixtures.The solutions of compound 3a in acetonitrile are pale yellow.With the addition of the anions, a change in the color of the solutions, from pale yellow to blue, was observed for CN -and F -, and with less intensity for CH3COO -and H2PO4 -(Figure 1A).This is exactly the coloration of the solution obtained from the deprotonation of the compound with the use of tetra-nbutylammonium hydroxide, which demonstrates that these anions abstract the proton of the phenolic moiety in the chemosensor, according to their basicity, to generate its deprotonated form, i.e., 3b species.It is still possible, with the naked-eye analysis of Figure 1A, to verify that, considering the same concentration, the less basic anions CH3COO -and H2PO4 -were less able to abstract the proton of 3a, in comparison with the more basic anions F -and CN -.None of the other anions caused a change in the visual aspect of the 3a solution.
Small amounts of water were added to the system, aiming to obtain selectivity towards one species of the anions studied.No changes in the color of the solutions of 3a were observed in the system containing H2PO4 -and CH3COO -(Figure 1B).With 2.4% of water present in solution, only CN -was found to be sufficiently basic to form 3b (Figure 1C).
Figure 2 shows the UV-vis spectra for compound 3a in acetonitrile and in acetonitrile with small amounts of water.This compound in its deprotonated form 3b exhibits a Vis band in acetonitrile with a max of 592 nm.The effect of the addition of anionic species on the UV-vis spectra of 3a in acetonitrile was then studied.The UV-vis spectrum for the solution of 3a with F -added shows a Vis band with the maximum absorbance coinciding with that obtained for the phenolate 3b, which was obtained with an excess of tetra-n-butylammonium hydroxide.A similar observation can be made with the solution of 3a in the presence of CN -, but with a slightly less intense effect, followed by CH3COO -and, with least intensity, H2PO4 -.In other words, the data demonstrate that in acetonitrile the ability of the anions to abstract the proton of 3a and generate 3b observes the following increasing order: H2PO4 -< CH3COO -< CN -< F -. None of the other anions were able to deprotonate the compound in solution, nor did they show the appearance of a Vis band in a region that would indicate the formation of species formed by HB.
Figure 2 also shows the influence of water on the UV-vis spectra of 3a in the presence of the anions.With the addition of only 0.5 % (v/v) of water H2PO4 -does not cause any spectral effect while under the same conditions CN -becomes a more efficient base than F -.It can be also observed that in the presence of 1.0% (v/v) of water CH3COO -is no longer detected.With the addition of 2.4% (v/v) of water to the solution, selectivity for CN -in relation to the other anions was achieved, due to the strong preferential solvation of F -, CH3COO -, and H2PO4 -for water through HB, inhibiting them from acting efficiently as bases.In addition, the presence of 0.5%, 1.0%, and 2.4% (v/v) of water in the solutions of 3b in acetonitrile led to a hypsochromic shift of 15, 27, and 43 nm, respectively, in the position of the Vis band.This indicates that 3b exhibits a very important negative solvatochromism and that in mixtures of acetonitrile with very small amounts of water the dye is preferentially solvated by water, more specifically by means of HB involving water molecules and the phenolate moiety of 3b.This kind of interaction results in a hypsochromic shift of the solvatochromic band of the compound in comparison with the position of the band in pure acetonitrile.These results are consistent with data reported in the literature for dyes with a phenolate group in their structure, such as compounds 1 and 2. 16 The results discussed in the previous paragraph can be better visualized in Figure 3, which shows that the selectivity for CN -, in relation to the other anions, is gradually reached with the addition of water to the system.The highest value for the absorbance corresponding to the max value was verified for F -in acetonitrile, but CN -gave the most important spectral effect with the addition of only 0.5% (v/v) of water, while with the presence of 2.4% (v/v) of water only CN - was able to modify the spectrum of the chemosensor in solution.), added to the solutions of 3a in (A) acetonitrile and in acetonitrile with (B) 0.5%, (C) 1.2%, and (D) 2.4% of water.The concentrations of 3a and of the anions are given in Figure 1.

Titration of 3a with the anions
Compound 3a was titrated with the anionic species able to change the color of the solutions in acetonitrile and in acetonitrile-water mixtures.The absorbance values for the maximum of the Vis band of 3b in acetonitrile and in each mixture were plotted as a function of the anion concentration added.The experimental data were fitted with the use of Eqs.1-3, 10,11,17 which are related to the following situations according to different chemosensor:anion stoichiometries: Case 2. 1:1 and 1:2 stoichiometries Case 3.
In these equations, Abs is the absorbance value after each addition of the anion, Abs0 is the initial absorbance without anion added, Abs11 and Abs12 are the maximum absorbance values obtained by addition of the anion considering 1:1 and 1:2 3a:anion stoichiometries, CA-is the anion concentration in each addition, and K11 and K12 are the binding constants.The results are given in Table 1 and show very good fits for all systems studied (S.D. < 1.0×10 -4 ).  4 shows the titration of 3a with F -in acetonitrile.It can be seen that with the addition of the anion a band with maximum absorbance at 592 nm appears, relating to 3b.A plot of the absorbance values at 592 nm as a function of the concentration of F -(Figure 5A) showed a sigmoidal shape, with a behavior typical of a 1:2 chemosensor:anion stoichiometry and the fitting of the experimental data led to a binding constant of K12 = (1.27±0.03)×10 8dm 6 mol -2 (Table 1).In Figure 5B the Job's plot is shown, which confirms 1:2 3a:anion stoichiometry.The final concentration of F -was 2.0×10 -4 mol dm -3 and the absorbance values were collected at 592 nm.(B) Job's plot for compound 3a with F -in acetonitrile.
The addition of very small amounts of water led to a change in the profile of the titration with F -, and this is due to a strong interaction of water with the anion, hindering its interactions as a base.A change in the 3a:anion stoichiometry occurred with the addition of 1.0% (v/v) of water to the system containing 3a from 1:2 to both 1:1 and 1:2 (Table 1).
The titration of 3a in acetonitrile with increasing amounts of CN -was made.It could be observed that the band with a max value of 378 nm related to compound 3a shows a reduction in the absorbance with the addition of the anion, and simultaneously a band with maximum absorbance at 592 nm, due to the appearance of 3b, occurs with an isosbestic point at 403 nm.The corresponding titration curve was obtained with experimental data for the absorbances at 592 nm and these data were fitted using Eq. 3, giving the values of K11 = (9.88±1.33)×10 3 dm 3 mol -1 and K12 = (7.58±0.63)×10 4 dm 6 mol -2 (Table 1).The addition of water to the system had a considerable effect not only on the magnitude of the binding constants but also on the profile of the process: with the addition of 5% (v/v) of water the stoichiometry observed was 1:1 3a:anion, with K11=(1.29±0.08)×10 3 dm 3 mol -1 .
The titrations with CH3COO -in acetonitrile had a behavior similar to those observed for CN - , with 1:1 and 1:2 3a:anion stoichiometries, changing to 1:1 with the addition of 0.5% (v/v) of water to the system.No effect of H2PO4 -was observed on the solution containing 3a in acetonitrile with 0.5% (v/v) of water, although the behavior for this anion in acetonitrile without water added is similar to that observed for CN -and CH3COO -.The results are clearly due to the effect of water solvating the species in solution (Table 1).

The role of water on the behavior of 3a in the presence of the anions
The titrations of 3a with the anions show two possible 3a:anion stoichiometries, 1:1 and 1:2, according to Scheme 1.If the stoichiometry is 1:1, the proton can be transferred directly from 3a to the anion.The other possibility involves firstly the interaction of the anion with the phenol moiety of the chemosensor through HB, and this interaction weakens the O-H bond.After this, a second equivalent of the anion is needed to abstract the proton, with the formation of complex species of the type [HA2] -.This has been reported in other papers 2i,10,18 and the formation and the stability of [HA2] -complexes, such as [HF2] -, have been studied by means of theoretical calculations.).20  The strong solvation of the anions by water makes the hydrated species less able to act as a base.Since CNis less hydrated with the addition of water, this anion becomes more 'naked' in relation to its ability to act as a base, being more efficient in the abstraction of the proton of 3a.The effectiveness of the use of water as a strategy to increase the selectivity of the chemosensor for CNover other more hydrated anions has been reported in the recent literature.2i, 10,11,21  The addition of water has another important role, which is to promote the stabilization of the conjugated base 3b through HB, making 3a more acidic.This explains the change observed in the 3a: anion stoichiometry (Table 1), which can be observed through the change in the shape of the titration curves, as showed in Figure 6 for the titrations of 3a using CNin acetonitrile and in its mixtures with small amounts of water.The titration curve of 3a with CNin acetonitrile has a sigmoidal shape, typical of 1:2 stoichiometry, which is gradually replaced by a curve with a shape typical of 1:1 stoichiometry when up to 6% (v/v) of water is added to the system.
Figure 7 shows the influence of water on the binding constants obtained from the titration curves of 3a with CN -(Table 1).The addition of water caused a very important decrease in the K12 values and, curiously, the K11 values increase with the addition of up to 2.5% (v/v) of water and then decrease above this water content.Since the presence of small amounts of water to the medium makes 3a more acidic, this explains the increase in the K11 values with the addition of up to 2.5% (v/v) of water in the system containing 3a and CN -.Another important aspect is that with the addition of water to the system the polarity of the medium increases and the selectivity for CNis achieved due to its lower hydration energy in comparison with the other anions.
Furthermore, water is able to solvate the anion and 3b, hindering the interaction between the compound and the anion, being responsible for a strong solvation of the anion, causing a considerable weakening in its action as a base and consequently lowering the values of binding constants.This makes the design of chemosensors for F -in aqueous medium a challenging and very difficult task.

Conclusions
Compound 3a was demonstrated to be an efficient anionic chromogenic chemosensor based on a simple acid-base strategy.In acetonitrile, the following increasing order was obtained: H2PO4 -< CH3COO -< CN -< F -, which matches the increasing order of basicity for these anions, and for the other anions studied no spectral changes were verified.The selectivity for CN -in relation to all other anions studied was achieved with the use of only 2.4% (v/v) of water due to the fact that this solvent, or hydrogen-bonded acetonitrile-water complexes, can preferentially solvate anions such as H2PO4 -, CH3COO -, and F -through HB, leading CN -freer to interact with the chemosensor.
The titrations of 3a with the anions that caused alterations in its UV-vis spectrum revealed the occurrence of two chemosensor:anion stoichiometries.The anion can abstract the proton from the chemosensor in one step, in a 1:1 stoichiometry, or in two steps, with the formation of [HA2] -complexes, in a 1:2 stoichiometry.The addition of water causes a decrease in the K12 values because the hydroxylic solvent interacts with both the anion and the phenol moiety of 3a, making the interaction between the anion and the compound more difficult.In addition, water is able to stabilize through HB the conjugated base of the chemosensor, making it more acidic, and making the anion able to abstract the proton in one step.
For the chemosensors with a pyridinium cation as an acceptor center in their molecular structure, as is the case of the merocyanines 1 and 2, titrations with basic anions in acetonitrile lead to a 1:3 chemosensor:anion stoichiometry, due to an electrostatic interaction between the pyridinium group and the anion prior to the interaction of the anion with the phenol moiety in the compound. 10For a compound with a more simple molecular structure, without a cationic center in its structure, such as 3a in acetonitrile with CN -, 1:1 and 1:2 3a:anion stoichiometries are observed, while for F -the stoichiometry observed is only of type 1:2.Therefore, the stoichiometry obtained is dependent on the molecular structure of the chemosensor, of the medium, and of the anion used.
Compound 3a was prepared in two steps.Firstly, 4-amino-2,6-diphenylphenol was obtained according to the procedure described in the literature, 23 for which 2,6-diphenylphenol (Aldrich) was stirred with an excess of sodium nitrite followed by the reduction of the product with granulated tin and concentrated HCl (yield: 59.7%; p.f. obs.138 °C (p.f.lit. 23135-138 °C); IR max/cm -1 (KBr): 3510 (O-H, m), 3427 (NH2, m), 3060, 2872 (=C-H, m), 1423, 1470, 1595 (C=C, m), and 1342 (C-N, s).This amine was used to prepare 3a through the following procedure: 4-nitrobenzaldehyde (0.24 g, 1.6 mmol), 4-amino-2,6-diphenylphenol (0.50 g, 1.9 mmol), and ethanol (15 mL) were added to an Erlenmeyer flask.The contents of the flask were then heated slowly with stirring until the complete solubilization of the reactants.One drop of acetic acid was then added and the reaction mixture was stirred for four hours.The mixture was then left to stand in a freezer overnight.The mixture was poured into cold water with stirring and the solid obtained was filtered under vacuum, washed with iced ethanol and recrystallized three times from methanol.The product, after drying, was an amorphous yellow solid (yield of 16.0 %), with a melting point of 134.7 ºC.IR (KBr, max/cm -1 ): 3539 (O-H, m), 1588 (C=N, m), 1475, 1425 (C=C, m), 1518, 1344 (N=O, vs), and1227 (C-O, s). 1  UV-vis studies of 3a with the anions UV-vis measurements were performed with a Varian Cary Bio 50 spectrophotometer at 25 °C, using a 1 cm quartz square cuvette.The maxima of the UV-vis spectra (max) were calculated from the first derivative of the absorption spectrum.A solution of 3a was prepared in acetonitrile in a concentration of 5.9×10 -5 mol dm -3 .This solution was then used to prepare the solution of each anion in a concentration of 6.0×10 -4 mol dm -3 , using 5 cm 3 volumetric flasks.Subsequently, these solutions were transferred to cuvettes hermetically closed with rubber stoppers in order to minimize the evaporation of the solvent and to avoid the entrance of water to the system and the UV-vis spectra were taken.All experiments were carried out at 25 °C.The experiments involving acetonitrile-water mixtures were carried out using the following procedure.Solutions of 3a containing each anion were prepared in acetonitrile, as described in the previous paragraph.Small volumes of water were then added to each system that gave a positive response, that is, a change in the color of the acetonitrile, and UV-vis spectra were taken in order to evaluate whether this amount was sufficient to allow selective detection with the naked-eye.The absorbance values were collected for the max values verified in each mixture.

Titration experiments
Titration experiments in acetonitrile were performed with the preparation of the solution of 3a as described previously.This solution was used to prepare the stock anion solutions in flasks closed with rubber stoppers and the titrations were carried out by adding small amounts (2-10 L) of the salt solution with a microsyringe to closed quartz cuvettes containing the solution of 3a.The UV-vis spectra were taken after each addition and the absorbance values were collected at 592 nm.Titration experiments were also performed in acetonitrile-water systems, using the minimal water content which allowed selective detection of the anion, defined in the previous UV-vis studies.

Stoichiometry determinations
The experiment for the stoichiometry determination was performed in acetonitrile with the method of continuous variations (Job's method).17a These plots were obtained considering the formation of 3b produced from 3a and the anionic species.Thus, these studies were performed at 25 °C using stock solutions in the experiment with concentrations of 6.0×10 -5 mol dm -3 for 3a and 7.0×10 -5 mol dm -3 for F -and the absorbance values were read at max=592 nm. d,e,g,h,i,j,k)

Scheme 1 .
Scheme 1. Possible interactions of 3a with the anions in acetonitrile.