Exploring urea–fluoride interactions in the vicinity of a tryptophan residue

Abstract

The effects of fluoride anions on tryptophan-derived isomeric urea ligands (UT1–UT3) were examined using UV-vis, fluorescence and ¹H NMR spectroscopy. Remarkably, the addition of TBAF triggered unusual enhancement of fluorescence for UT1, which emanated from intramolecular charge transfer following fluoride-induced deprotonation of the indole NH proton.

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Among the halides, the chemical and biological functions associated with fluoride anions are quite unique, yet complicated:¹ while it is recommended in dental care formulations, the exposure to excess fluoride is known to cause dental and skeletal fluorosis.² In biological systems, it has been suggested that accumulation of fluoride adversely affects biological signaling processes, inhibits certain enzyme functions and possibly reduces brain activity.³ Because of these factors, it is crucial to understand the nature of interactions of fluoride anions¹ with bioactive molecules such as indole and tryptophan. As such, aspects related to coordination and binding of fluoride has attracted immense attention, particularly in the context of anion recognition.⁴ Accordingly, several amide-based ligands containing the pyrrole, indole and pyridine motifs have been examined as receptors/sensors for fluoride anions.⁵ Again, the incorporation of urea and thiourea motifs can facilitate selective binding and recognition of the fluoride anion.⁶

In addition, colorimetric and fluorescent sensors based on indole and carbazole have been reported which respond to fluoride via anion-induced deprotonation.^5,6 Recent reports on fluoride recognition have illustrated various types of interactions between the indole ligand and fluoride, including anion-induce deprotonation of NH groups.^7–9 However, the nature of interactions between the urea motif and fluoride anions in the presence of tryptophan residues remains unexplored. Also, the discovery of haloalkane dehalogenase, wherein tryptophan coordinates to halide anions through the indole NH, was quite significant¹⁰ as it demonstrated hitherto unknown chemistry of this aromatic amino acid residue in anion recognition.

In an effort to understand how the presence of a tryptophan residue in close proximity could affect urea–fluoride interactions,¹¹ we synthesized and characterized isomeric tryptophan-derived urea ligands UT1–3 (Scheme 1) wherein the fluoride anion could potentially interact at the urea motif, the indole group or both. Earlier, the utility of urea-derived tryptophan ligands has been illustrated using N-substituted urea-derivatives of L-tryptophan which behave as antagonists for Neurokinin 1 (NK1) and Neurokinin 2 (NK2) receptors.¹²

Scheme 1 Structures of ligands UT1–UT3.

As shown in Scheme 2, the possible interactions between the indole and the urea groups for the ligands UT1–UT3 could be dependent on the ureido-benzamide ‘hinge’ group. Given the geometrical restraints of the ‘hinge’ group in UT1 and UT2, the tryptophan residues were expected to reside close to the urea motif as compared to UT3.

Scheme 2 Possible coordination of tryptophan during the interaction of fluoride anion and urea motif of UT2.

Thus, depending on the proximity of the pendant tryptophan residue to the urea motif, we hypothesized that coordination of fluoride to the ligands at the urea NH could also invoke participation of the indole NH groups (Scheme 2). Further, we anticipated that the interactions of fluoride with the ligands and the tryptophan residues could be monitored using absorption and fluorescence spectroscopy.

As shown in Fig. 1a, the UV-vis spectra of UT1 produced distinct absorptions at λ_max = 219 nm, 264 nm and weak band at 320 nm; in comparison, ligands UT2 and UT3 exhibited absorptions in the 270–300 nm range, as expected for tryptophan derivatives.¹³ Fig. 1b shows the UV-vis spectra recorded during the titration of UT1 with fluoride anions (TBAF) in acetonitrile. The addition of fluoride anions to UT1 caused the absorbance at 264 nm to gradually decrease, and concomitantly new absorption bands emerged at 283 and 357 nm (λ_max), along with an isobestic point at 272 nm (Fig. 2a). The red-shifted absorption at λ_max 357 nm was indicative of extended conjugation in the ligand system,¹⁴ caused apparently by the coordination of fluoride anion to UT1. For UT2, the UV-vis spectra revealed a gradual shift of the absorption at 258 nm to 271 nm (λ_max) upon addition of TBAF solution (Fig. 1c), and produced an isobestic point at 262 nm. Similarly, the addition of fluoride anions (>10 equiv.) to UT3 also caused shifting of the absorption at 268 nm (λ_max) to 291 nm, which was accompanied by the broad absorption at 350 nm (Fig. 1d). These changes in the ligand absorptions, and the associated bathochromic shifts observed upon addition of TBAF to UT1–UT3 were related to the formation of hydrogen bonded urea–fluoride complexes;^11,14 however, the role of the tryptophan pendant in the coordination of fluoride anion was not immediately understood.

Fig. 1 (a) UV-vis spectra of UT1–UT3 (conc. = 3.0 × 10⁻⁵ M in CH₃CN); Changes in the UV-vis spectra of (b) UT1 (c) UT2 (d) UT3 following the addition of fluoride anions (as TBAF in CH₃CN); (inset) plots of absorbances (UT1–UT3) vs. [TBAF] at the wavelength specified.

Fig. 2 Addition of TBAF to UT1 causes fluorescence enhancement at 438 nm (conc. 3.0 × 10⁻⁶ M, i.e. 0.003 mM in CH₃CN; λ_ex 295 nm); (inset) pictures of UT1 after addition of TBAF, taken in the dark under UV-illumination (365 nm) which show intense blue emission.

Tryptophan emissions are known to be environment-sensitive,¹⁵ a property which has been immensely informative for the study of protein structures and for probing enzyme activity.¹⁶ In this perspective, we undertook fluorimetric studies¹⁷ of UT1–UT3 with TBAF so as to elucidate the role of tryptophan in fluoride anion coordination.

As shown in Fig. 2, the addition of TBAF to UT1 in acetonitrile caused unexpected enhancement of fluorescence at 438 nm in place of the initial emission at 357 nm; the fluorescence variations at 357 nm and 438 nm as function of TBAF concentration has been shown in Fig. 2b. Notably, the emission of UT1 at 357 nm, which disappeared upon addition of TBAF, was coincident with the TBAF-induced absorption at 357 nm in the UV-vis spectrum. The red-shifted emission at 438 nm (Δλ_em 80 nm) was indicative of intra-molecular charge-transfer emissions,¹⁸ and possibly originated from the formation of UT1–fluoride complex¹⁹ (Scheme 3). Further, it was noted that the addition of protic solvents (e.g. methanol) affected the emission at 438 nm, while the addition of water (>25% v/v) caused quenching. In a competition experiment, we analysed the fluorescence profiles of the UT1–fluoride complex in the presence of chloride, bromide, iodide, acetate, nitrate, bicarbonate and hydrogen phosphate anions (upto 4.0 equiv., Fig S10†). It was revealed that the presence of acetate anions did not affect the fluorescence of the UT1–fluoride complex, while the addition of chloride, bromide, and iodide caused quenching of fluorescence.

Scheme 3 Proposed hydrogen bonding interactions in urea–fluoride complexes for UT1, UT2 and UT3, indicating the role of the indole groups (the ammonium cation not shown).

The fluorescence experiments revealed that the urea and indole groups of UT1 were significantly affected by fluoride coordination compared to UT2 and UT3. In fact, the incremental addition of TBAF to UT2 and UT3 caused quenching of fluorescence at 356 nm and at 374 nm respectively (Fig. S11†). Similar quenching of fluorescence could be observed for UT2 and UT3 in the presence of chloride, bromide, iodide, acetate, nitrate, bicarbonate and hydrogen phosphate anions.

As depicted in Scheme 3, we propose that the ligand geometry adopted by UT1 could be reminiscent of anthranilic foldamers featuring intra-molecular hydrogen bonding interactions.^20,21 This situation permits the indole motif of tryptophan (of UT1) to assist the formation of urea–fluoride complex, which in turn promotes deprotonation of indole NH groups.^7.8 As a result, intramolecular charge transfer (ICT) processes become favorable for UT1, which account for the changes observed in the absorbance and fluorescence spectra.^8,17 In contrast, the indole motifs in UT2 and UT3 would have restricted influence on the coordination of fluoride to the urea-motif; such variations in the nature of urea–fluoride complexation, with limited participation of the indole group could account for PET-induced quenching²² of ligand fluorescence in UT2 and UT3.

In order to substantiate the arguments made in Scheme 3, we examined the interactions of fluoride with UT1 in detail using ¹H NMR. As shown in Fig. 3, the addition of TBAF solution to UT1 caused distinct changes in the proton resonances corresponding to the indole and amide NH groups. The addition of 1.0 equiv. of TBAF ions to UT1 caused the deprotonation of the indole and urea NH groups, as indicated by the broadening and disappearance of the proton resonances at 10.03 and 9.32 ppm. The other urea NH resonance was broadened and shifted downfield from 9.23 ppm to 9.36 ppm. Further addition of TBAF (>2.0 equiv.) to the ligand was marked by the broadening of urea NH and the amide NH resonances at 9.77 ppm and 7.91 ppm respectively. Apart form these changes, the apparent deprotonation of the indole NH group produced a shielding effect, and consequently the resonances of the aromatic-CH groups shifted upfield reflecting increase of electron density in the phenyl rings.

Fig. 3 (a) Partial ¹H NMR of UT1 (4.3 mM in CD₃CN) showing the effect of TBAF on the indole NH (▲), urea NH resonances (●), amide NH (▼) and the aromatic CH (■) resonances at 25 °C.

Similar ¹H NMR titrations for UT2 and UT3 were performed in order to elucidate the nature of urea–fluoride interactions and the role of tryptophan residues vis-à-vis UT1. In both UT2 and UT3, distinct complexation-induced shifts emerged for the urea and indole NH groups, upon incremental addition of TBAF. The results indicated that both UT2 and UT3 could interact with two fluoride anions, invoking the urea and the indole NH groups.

The stoichiometry for UT2–F⁻ complex was determined using the Job's method (based on the urea NH resonances, Fig. 4), which indicated the formation of 1 : 1 complex at low anion concentrations; the apparent association constants (K_a)²³ was found to be 220(±24) M⁻¹. However, the Job's analysis for UT3–F⁻ system (as shown in Fig. S14†) revealed the formation of 2 : 1 complex, with apparent K_a 527(±30) and 75(±20) M⁻¹ respectively.

Fig. 4 (a) Partial ¹H NMR of UT2 (4.3 mM) following the addition of TBAF depicting the changes in the urea NH resonances (●), indole NH (▲), amide NH (▼) and the aromatic CH (■) resonances; (b) plots for urea NH resonances vs. [TBAF]; (c) Jobs plot for UT2–TBAF system indicates 1 : 1 complexation.

Conclusions

In conclusion, we have studied the nature of interactions of three isomeric urea-derivatives of tryptophan UT1–UT3 with fluoride anions using UV-vis, fluorescence and ¹H-NMR spectroscopy. We have shown that the addition of fluoride to UT1 produced unusual fluorescence enhancement apparently via ICT process whereas UT2 and UT3 exhibited quenching. On the basis of ¹H NMR evidence, we could establish that the observed changes in UV-vis and fluorescence spectra of ligand UT1 (compared to UT2 and UT3) were consequence of fluoride-induced deprotonation of indole NH groups. Under the same conditions, the addition of fluoride to UT2 and UT3 afforded distinct complexation-induced shifts to the urea and the indole NH resonances, however, without deprotonation. We have thus demonstrated a molecular system wherein the presence of tryptophan residue in close proximity dramatically affects the nature of urea–fluoride interactions, and promote deprotonation of the indole NH groups.

Acknowledgements

We gratefully acknowledge DAE-YSRA, Department of Science & Technology (DST) and UGC-DSA for funding & infrastructure facilities.

Notes and references

1. (a) M. Cametti and K. Rissanen, Chem. Soc. Rev., 2013, 42, 2016–2038; (b) P. A. Gale, Acc. Chem. Res., 2011, 44, 216–226; (c) J. L. Sessler, P. A. Gale and W. S. Cho, Anion Receptor Chemistry, RSC, Cambridge, UK, 2006, pp. 259–293; (d) K. L. Kirk, Biochemistry of the Halogens and Inorganic Halides, Plenum, New York, NY, 1991, pp. 58–86.
2. (a) E. Gazzano, L. Bergandi, C. Riganti, E. Aldieri, S. Doublier, C. Costamagna, A. Bosia and D. Ghigo, Curr. Med. Chem., 2010, 17, 2431–2441; (b) R. Marquis, E. S. A. Clock and M. Mota-Meira, FEMS Microbiol. Rev., 2003, 26, 493–510.
3. (a) J. L. Baker, N. Sudarsan, Z. Weinberg, A. Roth, R. B. Stockbridge and R. R. Breaker, Science, 2012, 335, 233–235; (b) Y. Yu, W. Yang, Z. Dong, C. Wan, J. Zhang, J. Liu, K. Xiao, Y. Huang and B. Lu, Fluoride, 2008, 41, 134–138; (c) S. X. Wang, Z. H. Wang, X. T. Cheng, J. Li, Z. P. Sang, X. D. Zhang, L. L. Han, X. Y. Qiao, Z. M. Wu and Z. Q. Wang, Environ. Health Perspect., 2006, 115, 643–647; (d) D. O'Hagan, J. Fluorine Chem., 2006, 127, 1479–1483; (e) D. O'Hagan and D. B. Harper, J. Fluorine Chem., 1999, 100, 127–133; (f) Y. Michigami, Y. Kuroda, K. Ueda and Y. Yamamoto, Anal. Chim. Acta, 1993, 274, 299–302; (g) R. J. Lesher, G. R. Bender and R. E. Marquis, Antimicrob. Agents Chemother., 1977, 12, 339–345.
4. (a) R. Sakai, A. Nagai, Y. Tago, S. Sato, Y. Nishimura, T. Arai, T. Satoh and T. Kakuchi, Macromolecules, 2012, 45, 4122–4127; (b) R. M. Duke and T. Gunnlaugsson, Tetrahedron Lett., 2011, 52, 1503–1505; (c) R. Perez-Ruiz, Y. Diaz, B. Goldfuss, D. Hertel, K. Meerholz and A. G. Griesbeck, Org. Biomol. Chem., 2009, 7, 3499–3504; (d) A. P. de Silva, H. Q. N. Guranatne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515–1566.
5. (a) M. Olivari, C. Caltagirone, A. Garau, F. Isaia, M. E. Light, V. Lippolis, R. Montisa and M. A. Scorciapinoa, New J. Chem., 2013, 37, 663–669; (b) X.-M. Liu, Q. Zhao, W.-C. Song and X.-H. Bu, Chem. – Eur. J., 2012, 18, 2806–2811; (c) G. W. Lee, K. Nam-Kyun and K. S. Jeong, Org. Lett., 2010, 12, 2634–2637; (d) D. Makuc, Triyanti, M. Albrecht, J. Plavec, K. Rissanen, A. Valkonen and C. A. Schalley, Eur. J. Org. Chem., 2009, 4854–4866; (e) D. Makuc, M. Lenarcic, G. W. Bates, P. A. Gale and J. Plavec, Org. Biomol. Chem., 2009, 7, 3505–3511; (f) P. A. Gale, Chem. Commun., 2008, 4525–4540; (g) F. M. Pfeffer, K. F. Lim and K. J. Sedgwick, Org. Biomol. Chem., 2007, 5, 1795–1799; (h) R. J. Sarma and J. B. Baruah, Cryst. Growth Des., 2007, 7, 989–1000; (i) G. W. Bates, P. A. Gale and M. E. Light, Chem. Commun., 2007, 2121–2123; (j) R. J. Sarma and J. B. Baruah, Chem. – Eur. J., 2006, 12, 4994–5000; (k) J. L. Sessler, D.-G. Cho and V. Lynch, J. Am. Chem. Soc., 2006, 128, 16518–16519.
6. (a) V. Amendola, G. Bergamaschi, M. Boiocchi, L. Fabbrizzi and L. Mosca, J. Am. Chem. Soc., 2013, 135, 6345–6355; (b) L. Pescatori, A. Arduini, A. Pochini, F. Ugozzoli and A. Secchi, Eur. J. Org. Chem., 2008, 109–120; (c) C. Caltagirone, J. R. Hiscock, M. B. Hursthouse, M. E. Light and P. A. Gale, Chem. – Eur. J., 2008, 14, 10236–10243; (d) D. Meshcheryakov, F. Arnaud-Neu, V. Bohmer, M. Bolte, J. Cavalieri, V. Hubscher-Bruder, I. Thondorf and S. Werner, Org. Biomol. Chem., 2008, 6, 3244–3255; (e) A. P. Davis, Coord. Chem. Rev., 2006, 250, 2939–2951; (f) D. R. Turner, M. J. Paterson and J. W. Steed, J. Org. Chem., 2006, 71, 1598–1608; (g) T. Gunnlaugsson, P. E. Kruger, P. Jensen, J. Tierney, H. D. P. Ali and G. M. Hussey, J. Org. Chem., 2005, 70, 10875–10878.
7. (a) A. Basu, S. K. Dey and G. Das, RSC Adv., 2013, 3, 6596–6605; (b) A.-F. Li, J.-H. Wang, F. Wang and Y.-B. Jiang, Chem. Soc. Rev., 2010, 39, 3729–3745; (c) V. Amendola, D. Esteban-Gomez, L. Fabbrizzi and M. Licchelli, Acc. Chem. Res., 2006, 39, 343–353.
8. (a) R. Sakai, N. Sakai, T. Satoh, W. Li, A. Zhang and T. Kakuchi, Macromolecules, 2011, 44, 4249–4257; (b) G. W. Lee, N.-K. Kim and K.-S. Jeong, Org. Lett., 2010, 12, 2634–2637; (c) R. Sakai, S. Okade, E. B. Barasa, R. Kakuchi, M. Ziabka, S. Umeda, K. Tsuda, T. Satoh and T. Kakuchi, Macromolecules, 2010, 43, 7406–7411; (d) H. M. Yeo, B. J. Ryu and K. C. Nam, Org. Lett., 2008, 10, 2931–2934.
9. (a) J. F. Zhang, C. S. Lim, S. Bhuniya, B. R. Cho and J. S. Kim, Org. Lett., 2011, 13, 1190–1193; (b) P. Bose and P. Ghosh, Chem. Commun., 2010, 46, 2962–2964; (c) Y. Shiraishi, H. Maeharaa and T. Hiraia, Org. Biomol. Chem., 2009, 7, 2072–2076; (d) Q.-S. Lu, L. Dong, J. Zhang, J. Li, L. Jiang, Y. Huang, S. Qin, C.-W. Hu and X.-Q. Yu, Org. Lett., 2009, 11, 669–672; (e) X. Y. Liu, D. R. Bai and S. Wang, Angew. Chem., Int. Ed., 2006, 45, 5475–5478.
10. (a) J. G. van Leeuwen, H. J. Wijma, R. J. Floor, J.-M. van der Laan and D. B. Janssen, ChemBioChem, 2012, 13, 137–148; (b) M. Pavlova, M. Klvana, Z. Prokop, R. Chaloupkova, P. Banas, M. Otyepka, R. C. Wade, M. Tsuda, Y. Nagata and J. Damborsky, Nat. Chem. Biol., 2009, 5, 727–733; (c) A. J. Oakley, M. Klvana, M. Otyepka, Y. Nagata, M. C. J. Wilce and J. Damborsky, Biochemistry, 2004, 43, 870–878; (d) L. Tang, A. E. J. van Merode, J. H. L. Spelberg, M. W. Fraaije and D. B. Janssen, Biochemistry, 2003, 42, 14057–14065.
11. P. Dydio, D. Lichosyst and J. Jurczak, Chem. Soc. Rev., 2011, 40, 2971–2985.
12. (a) D. A. Horton, R. Severinsen, M. Kofod-Hansen, G. T. Bourne and M. L. Smythe, J. Comb. Chem., 2005, 7, 421–435; (b) H. Qi, S. K. Shah, M. A. Cascieri, S. J. Sadowski and M. MaCcoss, Bioorg. Med. Chem. Lett., 1998, 8, 2259–2262; (c) A. M. Macleod, K. J. Merchant, F. Brookfield, F. Kelleher, G. Stevenson, A. P. Owens, C. J. Swain, M. A. Cascieri, S. Sadowski, E. Ber, C. D. Strader, D. E. Maclntyre, J. M. Metzger, R. G. Ball and R. Baker, J. Med. Chem., 1994, 37, 1269–1272.
13. (a) R. J. Sarma, A. K. Deka, S. Sinha and P. K. Bhattacharyya, J. Mol. Struct., 2013, 1052, 197–203; (b) C. Lincheneau, J. P. Leonard, T. McCabe and T. Gunnlaugsson, Chem. Commun., 2011, 47, 7119–7121; (c) H. Liu, W. Bao, H. Ding, J. Jang and G. Zou, J. Phys. Chem. B, 2010, 114, 12938–12947; (d) C.-Y. Wong and M. R. Eftink, Biochemistry, 1998, 37, 8938–8946.
14. C.-I. Lin, S. Selvi, J.-M. Fang, P.-T. Chou, C.-H. Lai and Y.-M. Cheng, J. Org. Chem., 2007, 72, 3537–3542.
15. (a) P. L. Muino and P. R. Callis, J. Phys. Chem. B, 2009, 113, 2572–2577; (b) P. R. Callis and T. Liu, J. Phys. Chem. B, 2004, 108, 4248–4259; (c) D. K. Smith and L. Muller, Chem. Commun., 1999, 1915–1916; (d) D. Porschke, C. Creminon, X. Cousin, C. Bon, J. L. Sussman and I. Silman, Biophys. J., 1996, 70, 1603–1608; (e) D. R. Ripoll, C. H. Faerman, P. H. Axelsen, I. Silman and J. L. Sussman, Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 5128–5132.
16. (a) I. Smirnova, V. Kasho, J. Sugihara and H. R. Kaback, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 8876–8881; (b) Y. Chen and H. P. Erickson, Biochemistry, 2011, 50, 4675–4684; (c) C.-P. Pan, P. L. Muino, M. D. Barkley and P. R. Callis, J. Phys. Chem. B, 2011, 115, 3245–3253; (d) M. Y. Berezin and S. Achilefu, Chem. Rev., 2010, 110, 2641–2684; (e) I. Smirnova, V. Kasho and H. R. Kaback, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 215661–221566; (f) H. Szmacinski, K. Ray and J. R. Lakowicz, Anal. Biochem., 2009, 385, 358–364; (g) J. Chen, S. L. Flaugh, P. R. Callis and J. King, Biochemistry, 2006, 45, 11552–11563; (h) S. W. Nelson, C. V. Iancu, J.-Y. Choe, R. B. Honzatko and H. J. Fromm, Biochemistry, 2000, 39, 11100–11106; (i) Y. Chen and M. D. Barkley, Biochemistry, 1998, 37, 9976–9982.
17. Emission profiles for UT2 and UT3 in the presence of TBAF provided as ESI.†.
18. (a) G. Baggi, M. Boiocchi, L. Fabbrizzi and L. Mosca, Chem. – Eur. J., 2011, 17, 9423–9439; (b) E. B. Veale and T. Gunnlaugsson, J. Org. Chem., 2008, 73, 8073–8076; (c) Y. Wu, X. Peng, J. Fan, S. Gao, M. Tian, J. Zhao and S. Sun, J. Org. Chem., 2007, 72, 62–70.
19. (a) Y. Shiraishi, H. Maehara, T. Sugii, D. Wang and T. Hirai, Tetrahedron Lett., 2009, 50, 4293–4296; (b) M. J. Chmielewski, L. Zhao, A. Brown, D. Curiel, M. R. Sambrook, A. L. Thompson, S. M. Santos, V. Felix, J. J. Davis and P. D. Beer, Chem. Commun., 2008, 3154–3156; (c) G. W. Bates, Triyanti, M. E. Light, M. Albrecht and P. A. Gale, J. Org. Chem., 2007, 72, 8921–8927; (d) K. J. Chang, B. N. Kang, M. H. Lee and K. S. Jeong, J. Am. Chem. Soc., 2005, 127, 12214–12215; (e) K.-J. Chang, D. Moon, M. S. Lah and K.-S. Jeong, Angew. Chem., Int. Ed., 2005, 44, 7926–7929.
20. T. Steiner, Angew. Chem., Int. Ed., 2002, 41, 48–76.
21. D.-W. Zhang, X. Zhao, J.-L. Hou and Z.-T. Li, Chem. Rev., 2012, 112, 5271–5316.
22. (a) G.-J. Zhao and K.-L. Han, Acc. Chem. Res., 2012, 45, 404–413; (b) J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, 2nd ed, Kluwer Academic/Plenum Publishers, New York, 1999, pp. 238–264.
23. H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703–2707.

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Footnote

† Electronic supplementary information (ESI) available: Details of synthesis, characterisation of UT1–UT3, along with fluorescence and ¹H NMR titration plots for UT2 and UT3 with fluoride. See DOI: 10.1039/c3ra46846a

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