Synthesis and Study in Solution of a New Dansyl-Modified Azacryptand

We report the synthesis of a new asymmetric azacryptand (L1), characterized by three p-xylyl spacers, one of which carries a dansyl side arm. The fluorescent sensor has been studied by potentiometric, UV-Vis, and emission studies in MeOH :water 3 : 2 mixture (0.07M NaNO 3 ), determining, in particular, the protonation constants of the free ligand and metal ion complexation equilibria. Interestingly, the obtained results revealed that the new receptor is fluorescent at neutral pH with a typical emission band of the dansyl group. Metal addition induced a partial quenching of the dansyl emission band; this behavior is more pronounced with Cu(II) that reduces the receptor’s emission by 60%. With all the studied cations, quenching follows the formation of a dimetallic complex. Similar studies on themodel compoundL2 confirmed that fluorescence quenching ismainly driven by a staticmechanism, attributable to the formation of the inclusion dicopper complex [L1Cu 2 ]. In order to test the stability of copper complexes under physiological conditions, spectrofluorimetric titrations with Cu(II) were performed in water buffered at pH = 8 (HEPES 0.07M) and the values of binding constants, K 11 and K 12 , were determined.

In general, fluorescent azacryptands show a weak fluorescence at neutral pH; in fact, amino groups are able to quench the fluorophore emission by a photoinduced electron transfer (PET) mechanism [26]. Among the best known fluorescent fragments, the dansyl fluorophore (5dimethylamino-1-naphthalene sulfonate) is characterized by an intense fluorescence emission in the visible spectrum, due to a charge-transfer excited state [27]. Moreover, high emission quantum yield and biological compatibility make the use of dansylated sensors very promising for application under physiological conditions [28][29][30]. In the present work, we report the synthesis and the characterization of the new asymmetric receptor containing a dansylated side arm (L1, see Scheme 1). In particular, the protonation constants of the free ligand and copper complexation equilibria in MeOH : water mixture have been investigated through potentiometric and spectrofluorometric experiments. The binding tendencies of the cage-like system towards copper (II) and

Synthesis of 2-Dansyl-1,4-benzenedicarboxaldehyde 2.
The dialdehyde 1 was obtained following the procedures reported in the literature [31]. Reagent 2 was prepared by modifying a described procedure [32]. 2-Hydroxy-1,4-benzenedicarboxaldehyde (0.1 g, 0.68 mmol) and dansyl chloride (0.2 g, 0.68 mmol) were suspended in 2 mL CH 2 Cl 2 . 0.1 g of 1,4-diazabicyclooctane (0.93 mmol) in 1 mL of CH 2 Cl 2 was added and the reaction mixture was stirred at room temperature for 48 hrs under an inert atmosphere. The crude product was purified by column chromatography (SiO 2 , hexane, hexane-AcOEt mixture 5%-25% gradient). A yellow solid was obtained (yield 52%). 1 [33]) in 120 mL of MeOH over 3 h at room temperature. After 24 h stirring, the mixture was heated to 50 ∘ C and hydrogenated with NaBH 4 (0.2 g, 5.2 mmol). When the addition was complete, the stirring was continued at 50 ∘ C overnight. The solvent was removed; the residue was dissolved in basic water (20 mL, 3 M NaOH) and extracted with 15 mL of CH 2 Cl 2 (×7). The collected organic phases were dried over Na 2 SO 4 and evaporated to dryness. An orange solid was obtained (yield 54%

Synthesis of L2.
In order to explain the coordination properties of the new fluorescent azacryptand, the model compound L2 was synthesized (Scheme 3). L2 was prepared by modifying a described procedure [32]: phenol (0.1 g, 1.0 mmol) and dansyl chloride (0.3 g, 1.0 mmol) were dissolved in 2 mL CH 2 Cl 2 . 0.2 g of 1,4-diazabicyclooctane (1.8 mmol) in 1 mL of CH 2 Cl 2 was added and the reaction mixture was stirred at room temperature for 24 hrs under an inert atmosphere. The crude product was purified by column chromatography (SiO 2 , hexane : AcOEt mixture 1 : 1). A yellow solid was obtained (yield 45%). 1   ligand solution was treated with an excess of a 1.0 M HNO 3 standard solution. Titrations were run by addition of 10 L aliquots of carbonate-free standard 0.1 M NaOH, recording 80-100 points for each titration. Complexation constants were determined by carrying out a similar potentiometric titration experiment, with the additional presence of 2 eqv. of Cu II (CF 3 SO 3 ) 2 . Prior to each potentiometric titration, the standard electrochemical potential (E ∘ ) of the glass electrode was determined in the MeOH/water (1 : 4) mixture, by a titration experiment according to the Gran method [34,35]. Protonation and complexation titration data (emf versus mL of NaOH) were processed with the Hyperquad5 package [36] to determine the equilibrium constants (reported in Table 1).
In the pH-spectrophotometric/spectrofluorimetric titration experiments, the UV-Vis/emission spectra of the solution were recorded after each addition of standard 0.1 M NaOH.

Spectrophotometric and Spectrofluorimetric Titrations.
UV-Vis spectra of the azacryptand in the presence of metal ions were run on a Varian Cary 100 SCAN spectrophotometer with quartz cuvettes of the appropriate path length (0.1-1 cm) at 25.0 ± 0.1 ∘ C under inert conditions. In any case, the concentration of the ligand and the optical path were adjusted to obtain spectra with AU ∼ 1.
Titrations with metal ions were performed at 25.0 ± 0.1 ∘ C in pure water and in MeOH/water (3 : 2) mixture, buffered at pH 8 (0.07 M HEPES). In a typical experiment, the solution of the receptor was titrated with a 100-fold more  3+ 10.63 (2) concentrated solution of the trifluoromethanesulfonate salt of the envisaged transition metals. In spectrofluorimetric titrations, the sample was excited at a wavelength corresponding to an isosbestic point in the family of UV-Vis spectra. Titration data were processed with a nonlinear least-squares procedure (Hyperquad package) [36], in order to determine the equilibrium constants.

Results and Discussion
3.1. Synthesis. Bistren symmetric cryptands are usually synthesized through Schiff base condensation of two molecules of tris(2-aminoethyl)amine (tren) and three molecules of the desired dialdehyde [37,38] followed by reduction with NaBH 4 of the imine groups. The new asymmetric azacryptand presented in this work required a multistep synthesis (see Scheme 2 in Section 2) recently described by our group [26,31,33]. In particular, 1 mol of mono-BOC-protected tren was first reacted with 1 mol of the 1,4-p-xylyl dialdehyde, obtaining the intermediate polyimine macrocycle 1. After reduction with NaBH 4 and deprotection of the secondary amine groups, precursor 1 was reacted with 2-dansyl-1,4benzenedicarboxaldehyde (2), in a 1 : 1 ratio. Reduction of the imine groups yielded L1 without further purification (see Section 2 for details). Thanks to this approach, we obtained a new fluorescent receptor with a fluorescent dansyl group covalently linked on one of the three p-xylyl spacers.

Potentiometric/pH-Spectrophotometric Studies in Aqueous
Mixture. In order to study the acid-base behavior of the new receptor, potentiometric titrations have been performed on the asymmetric azacryptand (L1) in H 2 O : MeOH 3 : 2 mixture (0.07 M NaNO 3 , at 25 ∘ C) by addition of standard acid. The use of a solvent mixture is fundamental to guarantee solubility in the whole pH range (2.5-11). Experimental results were fitted with Hyperquad package [36] and the obtained protonation constants are reported in Table 1.
In the pH range considered, only seven protonation constants could be determined, attributable to the protonation of the six secondary amines of the azacryptand and the tertiary amino group of the dansyl moiety. Notably, the protonation of the receptor's tertiary amino groups was not observed in these conditions. The obtained protonation constant of the NMe 2 groups, log = 1.7, is comparable to that of the system reported by Prodi and coworkers [39].
pH-spectrophotometric experiments confirmed these results; Figure 1 shows the family of spectra taken over the course of the pH-spectrophotometric titration of L1 (0.5 mM) in MeOH : H 2 O mixture (0.07 M NaNO 3 ).
At acidic pH values, the receptor shows the typical UV/Vis spectrum of the naphthyl group [40] with an intense band at max = 288 nm (black dashed line spectrum). Upon deprotonation of the dansyl moiety, the absorption at 288 nm decreases and a new band appears at 340 nm. This new band is attributable to the typical charge transfer of the dansyl group (NMe 2 → naphthyl) [27]. The titration profiles obtained at 288 and 340 nm well fit the distribution diagram of the species shown in Figure 2.
Analogous experiments have been carried out in the presence of 2 equiv. of Cu II (CF 3 SO 3 ) 2 . For cryptand L1, the best data fitting was obtained by assuming the formation of the following copper species on increasing pH: 3+ . The formation constants of the copper species are reported in Table 1 and the corresponding distribution diagrams are reported in Figure 3.
Between pH 2 and pH 4, the azacryptand is in the polyprotonated forms (i.e., L1H 7 7+ , L1H 6 6+ , and L1H 5 5+ ) in which both the secondary amino groups of the azacryptand and the dansyl tertiary amino group are protonated. On increasing pH, the formation of copper-containing species occurs: [CuL1H 3 ] 5+ (39% at pH = 4.5) and [CuL1H 2 ] 4+ (68% at pH = 5.1, see Figure 3). The dicopper complex, [Cu 2 L1] 4+ , is the major species in solution at pH 6.4 (78%). In [Cu 2 L1] 4+ , each Cu(II) ion occupies one of the tren units, adopting the typical trigonal bipyramidal geometry imposed by tripodal ligands. The apical positions on the metal ions, left free by the receptor, are occupied by water molecules. Upon further addition of NaOH, the deprotonation of   Table 1. Triangles: profiles of Mol Abs (×10 −3 , M −1 cm −1 ) at 288 nm (black) and 340 nm (white), respectively, obtained upon pH-spectrophotometric titration (right vertical axes, see Figure 1).  Table 1. Black triangles: profile of Mol Abs (×10 −3 , M −1 cm −1 ) at 300 nm versus pH, obtained upon pH-spectrophotometric titration of L1 (0.5 mM), in the presence of 2 equiv. Cu(CF 3 SO 3 ) 2 (right vertical axis, see Figure 4). coordinated water occurs, leading to the stable hydroxide complex [Cu 2 L1(OH)] 3+ . Due to its high stability, this species predominates in solution (>90%) over the wide pH range of 8.5-11. Figure 4 shows the family of spectra taken over the course of the pH-spectrophotometric titration of L1 (0.5 mM) in the presence of 2 eqv. Cu(CF 3 SO 3 ) 2 . The formation of copper-containing species is accompanied by the development of a band around 300 nm, attributable to a ligand to metal charge transfer (i.e., N(amine) → Cu(II)). The LMCT band develops at pH = 3.5 with the formation of the monometallic complex [CuL1H 3 ] 5+ and reaches its maximum intensity at pH = 6.8, in the presence of the dicopper species [Cu 2 L1] 4+ . Upon deprotonation of the metal coordinated water molecule, the charge-transfer band undergoes a slight blue shift (see Figure 4 solid black spectrum) due to the different coordinative sphere of the copper(II) cation. The profile obtained at 300 nm well fits the distribution diagram of the Cu(II) species shown in Figure 3.

pH Spectrofluorimetric Studies in Aqueous Mixture.
Thanks to the presence of the dansyl fragment close to the receptor's cavity, the formation of copper-containing species could be followed by emission studies. The fluorescence emission of L1 is pH dependent; therefore, pH spectrofluorimetric experiments on the asymmetric azacryptand (L1) were performed in an H 2 O : MeOH 3 : 2 mixture (0.07 M NaNO 3 , at 25 ∘ C) by addition of standard NaOH 0.1 M. Emission spectra were recorded after each addition of standard 0.1 M NaOH; the excitation wavelength was chosen at 314 nm, corresponding to an isosbestic point in the pHspectrophotometric titration (see Figure 1). The family of the emission spectra taken over the course of the titration of L1 (0.05 mM) is reported in Figure 5.
Noticeably, on increasing pH, a fluorescent band centered at 500 nm develops; this behavior is attributable to the formation of a CT band following the deprotonation of the dansyl tertiary amino group [41,42]. The observed emission band is typical of the dansyl fluorophore, characterized by an intense broad luminescence band.
Generally, the emission intensity of fluorescent polyammonium azacryptands decreases with pH; as an example, our group recently reported a fluorescent azacryptand with a 9,10-anthracenyl unit embedded in the receptor's framework [26]. The anthracenyl emission intensity was maximum at acidic pH (2 < pH < 4 interval) when the secondary amino groups were fully protonated. At pH > 4, the deprotonation of the ammonium groups occurred, leading to the quenching of the anthracenyl fluorophore by a photoinduced electron transfer mechanism, from the free amine groups to the excited anthracene. Very interestingly, in the case of L1, fluorescent emission increases with the pH, in particular between pH 3 and pH 5, reaching the maximum intensity over pH 7 (see Figure 6). The deprotonation of ammonium groups has no effect on the fluorophore emission because the dansyl group dangles out of the receptor's cavity, far from the amino groups. The photoinduced electron transfer is therefore disfavored by the distance between the fluorophore and the receptor's amines. Metal complexation generally affects the dansyl fluorescent band. Figure 7(a) shows the changes in the fluorescence of L1, observed over the course of the titration with copper. Cu(II) addition induced a partial quenching of dansyl emission (by about 60% of the initial intensity); the obtained titration profile / 0 at 538 nm is shown in Figure 7(d) (black triangles). Quenching follows the formation of a dicopper complex with a 2 : 1 Cu II : L stoichiometry; in fact, the quenching profile reaches a plateau upon the addition of 2 eqv. Cu(II). Receptor L1 can include two Cu II ions, one for each tren subunit. The encapsulated Cu(II) metal centers (d 9 ) easily quench the excited state of the fluorophore through a static quenching mechanism (electron transfer or energy transfer process). In order to prove that receptor's quenching was due to the formation of a stable dicopper complex, emission experiments were also performed on the model compound L2 (Figure 7(c)). L2 shows the typical band of the dansyl fluorophore. Notably, the addition of Cu(II) to a solution of L2 had no effect on the emission (even at a Cu(II) concentration 5-fold higher than L2; white triangles in Figure 7(d)). This result confirms that, in the case of L1, fluorescence quenching is manly driven by a static mechanism, associated with the coordination of copper ions within the receptor's cavity.

Studies on
Binding studies on L1 were performed also with Zn(II) in the same experimental conditions. Upon addition of zinc, the emission band of L1 undergoes a blue shift and a small decrease in intensity (Figure 7(b)). The titration profile (normalized intensity at 540 nm versus equivalents of Zn(II)) in Figure 7  if zinc has no redox properties, the coordination of Zn(II) induces a weak decrease of the fluorescence emission, indicating the existence of a chelating enhancement of the quenching [30]. In order to evaluate the stability of the copper complex in physiological conditions, complexation was also investigated in pure water. In particular, spectrofluorimetric titrations were performed on L1 (0.015 mM) with aqueous Cu(II) at pH = 8 (HEPES), where the [Cu 2 L1(OH)] 3+ complex is the predominant species (85%) in solution. Figure 8(a) displays the family of emission spectra recorded over the course of the titration L1 (0.015 M), adjusted to pH 8 with HEPES buffer (0.07 M), with a solution of 2.5 mM of Cu(CF 3 SO 3 ) 2 . On copper addition, the receptor's fluorescence is progressively quenched (by about 40% of the initial intensity), proving the interaction of L1 with Cu(II). The best fitting of the experimental data was obtained by assuming the formation of two species in solution with 1 : 1 and 2 : 1 Cu II : L stoichiometry; the corresponding binding constants are reported in Table 2. Figure 8(b) shows the distribution diagram of the species obtained from the values of K 11 and K 12 reported in Table 2. The emission intensity profile versus eqv. of Cu(II) (Figure 8(b)) well fits the concentration profile of the free ligand (solid line). Noticeably, the formation of the dicopper complex causes a partial quenching of the dansyl emission band.

Conclusions
In this work, we report the synthesis of the new asymmetric fluorescent azacryptand L1, characterized by a dansylated fragment anchored to one of the p-xylyl spacers. The sensor was fully characterized by potentiometric, UV-Vis, and emission studies in MeOH : water 3 : 2 mixture (0.07 M NaNO 3 ). Interestingly, the obtained results revealed that the new receptor is fully fluorescent at neutral pH, with the typical dansyl emission band. Notably, fluorescent azacryptands are usually quenched at neutral pH due to the photoinduced electron transfer (PET) from amino groups to the fluorophore. The binding tendencies of L1 towards copper (II) and zinc (II) were studied in MeOH and aqueous solution. Metal addition induced a partial quenching of L1's emission; this behavior was more pronounced with Cu(II), which reduces the emission intensity by about 60%. The profiles obtained in these studies with metal ions pointed out the formation of a dimetallic species with a 2 : 1 M II :L stoichiometry. Studies on the model fluorophore L2 confirmed that, in the case of L1, the quenching is mainly driven by the formation of an inclusion complex. The coordination properties towards Cu 2+ have been also tested under physiological conditions. Spectrofluorimetric titrations in water at pH = 8 with Cu 2+ allowed for the determination of the formation constants of both mono-and dicopper complexes.