A new fluorescent chemosensor for fluoride anion based on a pyrrole–isoxazole derivative

Molecules containing polarized NH fragments that behave as anion-binding motifs are widely used as receptors for recognition and sensing purposes in aprotic solvents. We present here a new example of a receptor, 3-amino-5-(4,5,6,7-tetrahydro-1H-indol-2-yl)isoxazole-4-carboxamide (receptor 1), which contains pyrrole, amide and amino subunits. This receptor shows both changes in its UV–vis absorption and fluorescence emission spectra upon the addition of F−, resulting in highly selectivity for fluoride detection over other anions, such as Cl−, Br−, I−, HSO4−, H2PO4− and AcO− in CH3CN. 1H NMR titration, time-dependent density functional theory (TDDFT) calculations and other experiments confirm that the sensing process is brought about by deprotonation of the pyrrole-NH in receptor 1.


Introduction
The development of anion receptors has become a field of substantial interest and activity [1][2][3].Among the various artificial receptors reported in recent years, those employing polarized NH groups as anion-binding motifs have attracted considerable attention.Typical examples are charge neutral receptors containing pyrrole, amide, indolocarbazole, guanidium, imidazolium and urea/thiourea moieties.Usually, the anions are recognized via H-bonding, which is not easy to differentiate from deprotonation of protons on the receptor-NH [4,5].Some urea/thiourea-containing receptors could particularly recognize Y-shaped oxoanions by H-bonding and more basic anions such as fluoride by deprotonation.Fluoride is primarily used for prevention of dental caries [6], enamel demineralization while wearing orthodontic appliances, and in treatment of osteoporosis [7,8].However, excessive fluoride ingestion can cause skeletal and dental injuries, nephrotoxic changes in both humans and animals, and lead to urolithiasis.Hence, it is highly advantageous to develop high-effective sensors that can detect fluoride anion in food and animal feed.
In this work, we report a new fluoride receptor 1 (Figure 1), 3-amino-5-(4,5,6,7-tetrahydro-1H-indol-2-yl)isoxazole-4carboxamide [9], which contains receptive groups toward anions and no urea/thiourea moieties which avoids the problem of multi-anion sensitivity.The common anion recognition moieties, i.e., a pyrrole NH and an amide group, present in the structure behave as proton donors.Isoxazoles and their derivatives are important intermediates in preparation of many natural products and related compounds, and are used as antimicrobial antifungal and herbicide agents [10,11].Research on the mechanism of anion recognition is helpful for understanding the biological activities of pyrrole-isoxazole derivatives.The receptor 1 displayed an emission maximum at 400 nm with a fluorescence quantum yield of 0.067 (determined by comparison with 1,4-bis(5-phenyl-2-oxazolyl)benzene as the reference compound, similarly in all other cases) [12] when excited at 340 nm.The changes in fluorescence intensity of 1 upon the addition of particular anions are shown in Figure 3.This clearly shows that the fluorescence intensity was remarkably quenched and the emission peak red shifted from 400 nm to 432 nm upon the addition of F − , however no significant quenching was observed on the addition of other anions.The F − titration experiments were carried out in CH 3 CN for further investigation (Figure 5, Figure 6).With increasing F − concentration, the absorbance of 1 at 340 nm decreased significantly and a new adsorption peak appeared at 375 nm with a sharp isosbestic point formation at 352 nm, which indicated that only two species are present in the equilibrium throughout the titration process [14].The absorption peak at 375 nm in CH 3 CN was partially returned to 340 nm when a protic solvent such as methanol or water was introduced, which suggested that the interaction of 1 and F − is due to hydrogen bonding [15] or deprotonation [16].Because of the greater contribution of the electron density to the conjugated system in the deprotonated 1 it could be concluded that the interaction is deprotonation rather than the formation of hydrogen bond.Figure 6 shows the changes of fluorescence emission of 1 upon addition of F − in CH 3 CN where the emission maximum was red-shifted to 432 nm.With increasing F − concentration, the emission intensity was quenched by about 90% when 50 equiv of F − was added.The quantum yield of fluorescence was reduced to 0.031 in this case.The stoichiometry of the equilibrium was found to be 1:2 by the existence of the inflexion in the titration profile (insert) at 400 nm (Figure 7).Usually, the deprotonation of an NH moiety caused by F − includes two steps.The first step is the formation of a 1:1 stoichiometry host-guest complex through hydrogen bonding; the second step is the deprotonation of the host with the formation of 1 − anion and HF 2 − self-complex, as illustrated in equilibria 1 and 2: ( (2) The fluorescence intensity of 1 was not significant changed when less than 2 equiv of F − was added, which suggested the formation of hydrogen-bond complex of 1•F − .However, the fluorescence intensity decreased drastically on further increasing the F − concentration, which indicated that such a hydrogen-bond complex interacted further with F − in the formation of 1 − anion and HF 2 − .The stoichiometry of the total equilibrium could be determined by fitting the experiment data as being 1:2 between 1 and F − ; the same results were also obtained from the profile spectra.The stability constant of the two steps were obtained at the same time and were log K 1 = 2.58 ± 0.15 and log K 2 = 6.38 ± 0.15, respectively [17,18] (Figure 7).

Studies on reaction with hydroxide (OH − )
Tetrabutylammonium hydroxide was added to the solution of 1 in CH 3 CN to investigate the above process.Changes in fluorescence emission of 1 upon addition of F − and OH − in CH 3 CN were almost the same, except for the degree of quenching, as shown in Figure 8. Upon the addition of 5 equiv of OH − the fluorescence emission of receptor 1 displayed λ max at 408 nm and the intensity was quenched by about 51%.On the other hand, upon addition of F − the emission displayed λ max at 405 nm but the intensity was quenched by only about 33%, i.e., less than that caused by OH − .Considering the stronger basicity of OH − , it preferred to react with receptor 1 by deprotonation rather than by H-bonding [19].The similar phenomena upon the addition of F − and OH − suggested that receptor 1 recognizes F − in the same way as OH − .

H NMR titration
1 H NMR titration was also carried out to confirm the deprotonation of receptor 1 by F − .Figure 9 shows the series of 1 H NMR spectra of 1 upon the addition of increasing amounts of TBAF in DMSO-d 6 .As discussed above, HF 2 − anion was formed when the receptor was deprotonated by F − via a two-step process.We found from the 1 H NMR titration experiment that a new signal at 16.1 ppm (J HF = 120 Hz), which was attributed to the HF 2 − anion, appeared after 0.6 equiv of F − was added [20].
However, the HF 2 − anion could come from the deprotonation of either the NH group or of the solvent.However, the pyrrole CH proton signal (H 4 ) was upfield shifted with increasing F − concentration, reflecting the increase in electron density in the pyrrole ring [21].This supports the hypothesis that the F − preferably interacts with the receptor-NH rather than solvent molecules.Because of the higher charge density and smaller size, fluoride as strong base can deprotonate the receptor 1 to afford the heterocyclic conjugated anion [22,23], the emission of which is significantly lower than that of its charge neutral species when excited at 340 nm.
The deprotonation site could be determined by the 1 H NMR titration spectra (Figure 9) upon the addition of a sufficient amount of fluoride.There are three types of NH proton signals in the free receptor 1, which are designated as H 1 , H 2 and H 3 , respectively.It was found that the pyrrole NH (H 1 ) in the downfield part of the spectrum gradually broadened, weakened and finally disappeared with increasing F − concentration.Meanwhile, the signal for amide NH (H 3 ) also weakened and disappeared but gradually reappeared as two new signals at 6.5 ppm and 13.5 ppm after addition of 2.0 equiv of F − .However, the signal for the amino NH (H 2 ) was only slightly downfieldshifted (0.31 ppm) with no broadening or weakening during the same process.Obviously, the disappearance of the pyrrole NH (H 1 ) proton indicated clearly that the bifluoride signal at 16.1 ppm arose from the deprotonation of this NH moiety.

Time-dependent density functional theory (TDDFT) calculation
To further investigate the chemical transformation of receptor 1 from neutral to its anionic form, the lowest energy electronic excited states of receptor 1 and its potential anionic forms were calculated at the B3LYP/6-31G(d) level using the TDDFT approach on their previously optimized ground-state molecular geometries in CH 3 CN [24][25][26][27][28]. Wavelengths and the oscillator strengths are listed in Table 1.The absorption λ max of anionic  forms a and b (Figure 10) were calculated since the pyrrole NH and amide NH are typical proton donors which are both easily deprotonated by strong base [29].The calculated absorption wavelength of free receptor 1 was 338.5 nm, only 1.5 nm lower than the experimental value (340 nm) which suggests that TDDFT is suitable for calculating the absorption wavelengths of receptor 1 and its anionic forms.Thus the absorption wavelengths of its two anionic forms, a and b, were calculated to be 363.6 nm and 323.6 nm, respectively.The calculated absorption wavelength of anionic form a is much closer to the experimental result than that of anionic form b. Thus the NH fragment involved in the deprotonation process is the pyrrole NH rather than the amide NH.This conclusion is also in agreement with the results of the 1 H NMR titration experiment.

Conclusion
In conclusion, we report a new fluorescent chemosensor, pyrrole-isoxazole derivative, for fluoride recognition in CH 3 CN.The UV-vis and fluorescence titration experiments revealed that the receptor-NH could be easily deprotonated by fluoride via a two-step process.This is confirmed by the appearance of the HF 2 − anion in the 1 H NMR titration experiment, and the pyrrole-NH is considered to be involved in the deprotonation process.The result of time-dependent density functional theory calculation also indicates that the mechanism of anion recognition is via the deprotonation of the pyrrole-NH.
From UV-vis and fluorescence titration experiments it was found that the receptor 1 could recognize fluoride anion (F − ) with high selectivity and sensitivity over other anions (Cl − , Br − , I − , HSO 4 − , H 2 PO 4 − and AcO − ).Both 1 H NMR titration experiments and time-dependent density functional theory (TDDFT) calculations demonstrated that the mechanism is deprotonation of the pyrrole-NH.

Figure 4 :
Figure 4: Comparison of fluorescence emission of 1 (5 μM) in CH 3 CN after the addition of 50 equiv of tetrabutylammonium salts.

Figure 7 :
Figure 7:The fit of the experimental data of fluorescence emission of 1 (5 μM) upon the addition of F − at 400 nm to a 1:2 binding profile (excited at 340 nm).Inset: the partial enlarged curve when less than 5 equiv of F − was added.

Figure 10 :
Figure 10: Anionic form a and b of receptor 1.