Fluorescent calix[4]triazole for selective fluoride anion sensing

Abstract

Fluoride ions (F⁻) play an important role in preventing cavities and treating osteoporosis, but excessive exposure can lead to serious health problems such as fluorosis and kidney damage. These dual characteristics highlight the need for selective and sensitive methods to detect fluoride ions for health monitoring. Accordingly, in this study, we investigated the anion-binding ability of Py-CT4, a fluorescent chemosensor in which pyrene is linked to calix[4]triazole via an ester linker. Notably, Py-CT4 exhibited significant fluorescence quenching for F⁻ compared to other anions, and its fluorescence intensity gradually decreased with increasing F⁻ concentration. This phenomenon is driven by electron transfer from calix[4]triazole to pyrene, initiated by hydrogen bonding with F⁻ and followed by F⁻-induced deprotonation of calix[4]triazole. The selectivity of Py-CT4 for F⁻ appears to stem from its relatively flexible structure and low acidity compared to the previously reported Py-CT4+. Py-CT4 thus represents the first macrocyclic receptor based on charge-neutral 1,2,3-triazole that selectively recognizes F⁻ through fluorescence quenching. Compared to traditional detection methods, Py-CT4 utilizes the advantages of fluorescent detection, such as higher sensitivity, faster response times, and ease of use, for fluoride ion detection. Py-CT4 also demonstrates excellent selectivity for F⁻ even in the presence of competing anions. These features make Py-CT4 a promising tool for monitoring fluoride ions in biological and environmental systems, providing valuable insights into public health and safety.

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Introduction

The fluoride ion (F⁻) is the smallest anion, with a high charge density that confers unique chemical properties such as high electronegativity and strong basicity. Fluoride is a trace element essential for human health, and the body is highly sensitive to its concentration. Adequate fluoride intake has beneficial effects on dental health and osteoporosis treatment.¹ However, excessive fluoride exposure can lead to serious health issues, including fluorosis, urolithiasis, and cancer.² Therefore, monitoring fluoride is critical for health reasons. Among the various reported methods for detecting fluoride ions,³ fluorescence detection is a simple, cost-effective technique that enables rapid and highly sensitive monitoring. Accordingly, numerous fluorescent sensors for fluoride have been developed,⁴ most of which rely on intermolecular interactions such as hydrogen bonding and typically involve structures containing N–H and/or O–H hydrogen bond donors.⁵

Sufficiently polar C–H bonds can also act as hydrogen bond donors for anions.⁶ The 1,2,3-triazole moiety, with its high dipole moment, forms a polar C–H bond capable of binding anions via hydrogen bonding.⁷ As such, 1,2,3-triazole has become an important structural motif in the field of anion recognition.⁸ However, only one fluorescent chemosensor based on 1,2,3-triazole for fluoride detection has been reported to date, and its structure is limited to acyclic systems.⁹

The photoinduced electron transfer (PET) mechanism relies on fluorescence on/off switching and is widely used in anion fluorescent chemosensors.¹⁰ A typical PET-based fluorescent sensor follows a “receptor–spacer–fluorophore” design,¹¹ wherein analyte binding either inhibits or activates PET, producing or quenching fluorescence. Recently, various PET-based anion fluorescent chemosensors have been developed, in which receptors containing neutral hydrogen bond donors (e.g., amide, urea, or thiourea) are linked to fluorophores via spacers.¹² Most of these sensors exhibit fluorescence quenching upon anion addition, including fluoride.¹³ This is due to hydrogen bonding or deprotonation of the hydrogen bond donor by the anion, which increases the electron density of the donor and promotes electron transfer to the fluorophore. To the best of our knowledge, no PET-based fluorescent sensor using 1,2,3-triazole as a hydrogen bond donor has been reported that quenches fluorescence upon fluoride addition.

Our group previously developed macrocyclic calixtriazoles as a novel host platform, incorporating 1,2,3-triazole rings in place of the phenol moieties of calixarenes.¹⁴ Although some time has passed since the initial development of calix[n]triazole, its applicability has not been thoroughly explored. By contrast, the usefulness of its N-alkylated derivative, calix[n]triazolium, has been a main subject in our studies.

Several anion recognition studies using calix[n]triazolium have been conducted by our group. For instance, we successfully achieved selective sensing of adenosine monophosphate (AMP)¹⁵ and developed a crown ether-appended calixtriazolium system for recognizing H₂PO₄⁻.¹⁶ Additionally, we recently developed a PET fluorescent chemosensor functionalized with pyrene (Py-CT4+; Fig. 1A) and investigated its anion recognition ability.¹⁷ This chemosensor exhibited fluorescence quenching due to PET, caused by electron transfer from pyrene to electron-deficient calix[4]triazolium in the unbound state. When Py-CT4+ bound to HSO₄⁻, its fluorescence intensity increased due to the inhibition of PET.

Fig. 1 Our previous work and this work. (A) Previous work: calix[4]triazolium-based “off-on” fluorescent PET sensor. (B) This work: calix[4]triazole-based “on-off” fluorescent PET sensor.

In light of the several physicochemical differences between triazolium and triazole, we recently comprehended that the precursor for Py-CT4+, Py-CT4 (Fig. 1B), might also exhibit interesting and unique molecular sensing properties. The expected differences between Py-CT4+ and Py-CT4 were as follows: (1) the triazole C5-Hs of calix[4]triazole are less acidic than the triazolium C5-Hs of calix[4]triazolium.¹⁸ This suggests that calix[4]triazole can form a stable interaction with an anion that is more basic than the anion that binds to calix[4]triazolium. (2) In the ¹H NMR spectrum, calix[4]triazole in Py-CT4 appears as a single peak,^14a whereas calix[4]triazolium in Py-CT4+ shows a major single peak along with an additional minor peak (Fig. S1†).^15b This is presumably because calix[4]triazole has a more flexible structure that allows dynamic conformational changes. This flexibility may provide structural adaptation to other specific anions. (3) Neutral calixtriazoles have a higher electron density than positively charged calixtriazoles.¹⁹ Accordingly, when the calix[4]triazolium in Py-CT4+ is changed to calix[4]triazole, a change in the electron flow in the PET pathway is expected.

Based on these considerations, we describe here a study focused on identifying the anion-recognition ability of Py-CT4 (Fig. 1B). Specifically, this research explores the selective binding mechanism of Py-CT4, where the hydrogen bond-mediated interaction between calix[4]triazole of Py-CT4 and F⁻ activates PET, resulting in fluorescence quenching. This mechanism is due to the unique physicochemical properties of Py-CT4, which enable its selective recognition of F⁻ and the resulting “turn-off” fluorescence response. To the best of our knowledge, this is the first report of a PET-based macrocyclic “turn-off” fluorescent chemosensor that selectively recognizes anions through hydrogen bonding with 1,2,3-triazole.

Results and discussion

A fluorescence study was conducted to gain insight into the fluorescence properties of Py-CT4 for different anions (Fig. 2A and B). Py-CT4 exhibited turn-on fluorescence at 400 nm in its unbound state (Fig. 2A; see blank). Unlike Py-CT4+, in Py-CT4, the electron density of the calix[4]triazole is sufficiently high, making its energy level similar to that of pyrene. As a result, PET from calix[4]triazole to pyrene was thought to be suppressed, thereby maintaining the fluorescence of Py-CT4.²⁰ The binding ability of Py-CT4 to anions was investigated by analyzing the relative emission intensity changes of Py-CT4 after the addition of various anions (such as tetrabutylammonium salts, F⁻, Cl⁻, Br⁻, I⁻, H₂PO₄⁻, NO₃⁻, HSO₄⁻, or CH₃CO₂⁻) to Py-CT4. Most of the added anions had negligible effects on the fluorescence emission intensity of Py-CT4. In contrast, when the same concentration of fluoride anions was added to Py-CT4, quenched fluorescence was observed. This is because Py-CT4 has high flexibility, making it difficult to lock into the specific conformation required for binding with complex anions such as H₂PO₄⁻ and HSO₄⁻. As a result, Py-CT4 may exhibit low binding stability with such complex anions.²¹ By comparison, anions with simple structures, which require minimal conformational changes for binding, allow Py-CT4 to achieve greater stability, resulting in a tendency to prefer such anions.²² In addition, because Py-CT4 has very weak acidity, it prefers anions with high basicity for stable binding. Accordingly, Py-CT4 selectively recognizes F⁻, which has a simple structure and high basicity.²³ In the case of fluorescence quenching of Py-CT4 by F⁻, it appears to result from increased electron density of calix[4]triazole due to interaction with F⁻, which promotes PET from calix[4]triazole to pyrene.²⁴ These results clearly demonstrate that physicochemical properties of Py-CT4 influence its anion recognition and fluorescence response, leading to outcomes different from those of Py-CT4+.

Fig. 2 (A) Fluorescence spectra of Py-CT4 (10 μM, λ_ex = 355 nm) in the presence of 100 equiv. of various anions in a DMSO solution. (B) Corresponding fluorescence responses of Py-CT4 (10 μM) to different anions in a DMSO solution. (C) Fluorescence spectra of Py-CT4 (10 μM) upon titration with F⁻ (0–1000 equiv.) in a DMSO solution.

The sensing properties of Py-CT4 for F⁻ were further studied through fluorescence titration experiments (Fig. 2C). With the gradual addition of F⁻ to Py-CT4, the fluorescence intensity of Py-CT4 gradually decreased, indicating a close interaction between Py-CT4 and F⁻. These titration results were applied to Bindfit²⁵ to determine the stoichiometry and association constants between Py-CT4 and F⁻. Among BindFit's three binding models (1 : 1, 1 : 2, and 2 : 1), the 1 : 1 binding model with the least errors and uncertainties was the most appropriate, and the association constant between Py-CT4 and F⁻ was calculated as 74.30 ± 12 M⁻¹ (Fig. S2–S4 and Table S1†). Additionally, Job's plot shows a maximum at the 0.5 M fraction, supporting a 1 : 1 stoichiometric ratio of Py-CT4 and F⁻ (Fig. S5†).

Anion competition experiments were also performed to demonstrate the selectivity of Py-CT4 for F⁻. For this purpose, the change in the fluorescence intensity after the addition of F⁻ to a Py-CT4 solution containing interfering anions was recorded. As shown in Fig. 3, when equal amounts of F⁻ and competing anions (such as tetrabutylammonium salts, Cl⁻, Br⁻, I⁻, H₂PO₄⁻, NO₃⁻, HSO₄⁻, or CH₃CO₂⁻) were added to Py-CT4, the fluorescence intensity of Py-CT4 reduced by F⁻ was not significantly affected by the competing anions. Importantly, oxygen-containing anions, which are known to interfere with fluoride anion recognition, did not alter the emission intensity of Py-CT4 diminished by F⁻.²⁶ This indicates that Py-CT4 can be used as a selective fluorescent chemosensor for F⁻ in the presence of other competing anions.

Fig. 3 Fluorescence response at 403 nm of Py-CT4 (10 μM) containing 100 equiv. of F⁻ and selected anions (100 equiv.) in a DMSO solution (excitation at 355 nm).

To further investigate the binding interaction properties of Py-CT4 and F⁻, ¹H NMR titration experiments were performed (Fig. 4). Gradual addition of F⁻ to Py-CT4 caused the peak of the triazole proton (H_a) to become broader, decrease, and shift slightly downfield. When F⁻ was added in excess, the H_a signal eventually disappeared while a broad triplet peak was observed at 16.1 ppm, attributed to the proton of the hydrogen difluoride anion [HF₂]^–.^20a These results indicate that Py-CT4 forms C–H⋯F⁻ hydrogen bonds at low concentrations of F⁻, whereas deprotonation of the triazole proton occurs at higher concentrations of F⁻. This indirectly demonstrates that hydrogen bond donation by triazole to F⁻ plays an essential role in the selective recognition of F⁻ by Py-CT4. Along with the change in the triazole signal (H_a), most protons, except for the triazole proton, also showed a downfield shift. In particular, the protons located on the bridge between the triazole rings (H_b and H_c) showed a change from multiplet to singlet peaks along with a downfield shift, which indicates a structural change in the calix[4]triazole receptor framework.^14,27 This conformational change is presumably triggered by changes in the intramolecular hydrogen bonding environment of the calix[4]triazole receptor due to deprotonation of the triazole proton by F⁻.²⁸

Fig. 4 Partial ¹H NMR titration spectra of Py-CT4 (1.5 mM) in DMSO-d₆ with increasing concentrations of F⁻ anion.

Most ideal PET sensors have a spacer between the fluorophore and receptor, which prevents ground-state n–π or π–π interactions.²⁹ Accordingly, recognition of the analyte by the receptor is accompanied by no or only very small changes in the absorption spectrum of the fluorophore.³⁰ As shown in Fig. 5, the UV-Vis spectrum of Py-CT4 appears in the 250–400 nm range, and no noticeable change in absorption is observed upon the addition of F⁻ (100 equiv.). This result suggests that the ester spacer prevents interactions between calix[4]triazole and pyrene, indicating that there are no significant ground-state interactions between these two moieties. This provides indirect support that Py-CT4 is an ideal PET sensor.

Fig. 5 UV-Vis spectra of Py-CT4 (10 μM) in the presence of 0 or 100 equiv. of F⁻ in a DMSO solution.

Conclusions

This study explores the anion recognition properties of calix[4]triazole-based Py-CT4. In a previous study, Py-CT4+, a calix[4]triazolium-based fluorescent sensor, exhibited selective fluorescence turn-on due to PET inhibition when combined with the complex anion HSO₄⁻. In this study, we showed that calix[4]triazole-based Py-CT4 exhibits selective fluorescence quenching when combined with F⁻. The selectivity of Py-CT4 for F⁻ is believed to result from the combination of the high flexibility and low acidity of calix[4]triazole. Meanwhile, the fluorescence quenching of Py-CT4 toward F⁻ is expected to be caused by the facile PET from calix[4]triazole, whose electron density is increased by F⁻, to pyrene. These results indicate that Py-CT4 is a new neutral anion-sensing platform that exhibits anion selectivity and a fluorescence response different from that of charged Py-CT4+, through a combination of its unique physicochemical properties.

This study utilizes the different physicochemical properties of calix[4]triazole and calix[4]triazolium. Through this, we assess the potential of triazole and triazolium in the development of novel anion receptors. Additionally, F⁻ recognition by fluorescence changes of Py-CT4 presents a simple and cost-effective alternative to traditional analytical methods. Notably, the selectivity of Py-CT4 for F⁻ in the presence of competing anions suggests that Py-CT4 has the potential to lead to practical applications such as F⁻ detection in drinking water or biological samples. Future research will focus on optimizing the structure of calix[n]triazole to enhance its anion selectivity and sensitivity and utilize it for various applications such as biological diagnostics and environmental monitoring. Accordingly, these studies will provide new insights into anion recognition and become a useful technology for advancing medicine and the environment.

Data availability

The data used for this research is provided in the main article and its accompanying ESI.†

Author contributions

J. Cho performed all experiments and wrote the manuscript and ESI.† S. Kim supervised this study and revised the manuscript.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2023-00209322 and NRF-2018R1A5A2024425).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR spectrum and complexation studies between Py-CT4 and F⁻. See DOI: https://doi.org/10.1039/d5ra00014a

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