Amidofluorene-appended lower rim 1,3-diconjugate of calix[4]arene: synthesis, characterization and highly selective sensor for Cu2+

Functionalization of calix[4]arene with amidofluorene moieties at the lower rim led to formation of the 1,3-diconjugate of calix[4]arene L as a novel fluorescent chemosensor for Cu2+. The receptor molecule L exhibited a pronounced selectivity towards Cu2+ over other mono and divalent ions. The formation of the complex between L and Cu2+ was evaluated by absorption, fluorescence and 1H NMR spectroscopy. The sensor L showed a remarkable color change from colorless to purple and a fluorescence quenching only upon interaction with Cu2+. The 1:1 stoichiometry of the obtained complex has been determined by Job’s plot. The association constant determined by fluorescence titration was found to be 1.8 × 106 M−1. The sensor showed a linear response toward Cu2+ in the concentration range from 1 to 10 µM with a detection limit of 9.6 × 10−8 M.

Unlike Cu 2+ ions, the addition of 10 equiv of other metal ions to the 1.0 × 10 −5 M solutions of L resulted in no significant changes in the absorption spectra of ligand L. However, addition of Cu 2+ to L resulted in a blue shift of the absorption band in the area of 280-290 nm (see Supporting Information File 1, Figure S12). Furthermore, a weak and broad absorption band from 600-800 nm was also observed ( Figure 1). As shown in Figure 2, there was an evident color change from colorless to purple, which could be observed by the naked eye.
The selectivity of receptor L towards Cu 2+ was precisely assessed by UV-vis spectroscopy during titration with different concentrations of Cu 2+ from 0 to 100 equivalents in CH 3 CN. In the absence of Cu 2+ ions, the ligand L exhibited three absorption bands at ≈282, ≈290 and ≈314 nm ( Figure 3). Upon addition of Cu 2+ , the absorption band at 282 nm showed a blue shift along with an increase in peak intensity. The two observed isosbestic points at 295 and 318 nm upon addition of Cu 2+ revealed formation of a stable complex between L and the copper ion. Moreover, upon addition of Cu 2+ , at higher concentrations of L a broad signal appeared at the area of 600-800 nm which can be related to a d→d transition (see Supporting Information File 1, Figure S13). This result is in accordance with those reported in literature [10] and indicates that the L binds copper as Cu 2+ .

Fluorescence titrations of L with metal ions
Fluorescence spectroscopy was applied to investigate the recognition properties of chemosensor L toward various metal ions in MeCN. In the absence of metal ions, the receptor L represents fluorescence emission at ≈365 nm when excited at 280 nm. Upon addition 10 equiv of Cu 2+ to the MeCN solution of sensor L, a remarkable fluorescence quenching was observed ( Figure 4). In order to explore the selectivity of sensor L, simi-    lar experiments were carried out in the presence of other perchlorate salts of Cu 2+ , Hg 2+ , Pb 2+ , Zn 2+ , Co 2+ , Ni 2+ , Cd 2+ , Ag + , Ba 2+ , K + , Na + and Li + . The fluorescence spectra showed almost no obvious change relative to the free ligand L ( Figure 4).
Titration of L with Cu 2+ resulted in a gradual quenching of the fluorescence emission at the 365 nm band as a function of increasing Cu 2+ concentration. The fluorescence intensity spectrum showed a gradual decrease up to 5 equiv of Cu 2+ . With higher Cu 2+ concentrations the fluorescence quenched completely ( Figure 5a). The stoichiometry of the complex was found to be 1:1 between L and Cu 2+ based on the Job's plot and its association constant was calculated using the Benesi-Hildebrand equation [35] to be 1.8 × 10 6 M −1 (see Supporting Infor-   mation File 1, Figures S14 and S15). From these results it can be concluded that the Cu 2+ ion has a preferential interaction with receptor L. It can be concluded from the results displayed in Figure 5b that the fluorescence quenching intensity at 365 nm has a linear relationship with Cu 2+ concentration. The fluorescent response of L toward Cu 2+ has a linear dynamic range from 1 to 10 µM with a detection limit of 9.6 × 10 −8 M (see Supporting Information File 1, Figure S16).

Competitive metal ion titrations
In order to examine the competitive recognition of Cu 2+ by sensor L, the effect of Cu 2+ was studied in the presence of other metal ions. The results showed that the fluorescence emission intensity of [L + M n+ ] is altered in the presence of Cu 2+ ions ( Figure 6). It is noteworthy that complex [L + M n+ ] exhibited no considerable fluorescence variations in comparison with L, and addition of Cu 2+ ions to the corresponding solutions resulted in fluorescence quenching. Therefore, L can recognize Cu 2+ even in the presence of other metal ions in acetonitrile solution.

H NMR titration of L with Cu 2+
In order to understand the mode of complexation of L with Cu 2+ , 1 H NMR titrations were carried out (Figure 7). Upon the initial addition of Cu 2+ into the CD 3 CN solution of L, signals related to OH phenolic groups (Hb) and amidic protons (Ha) were first broadened and then finally disappeared. Furthermore, the signals of Ar-CH 2 (Hd, He) and -O-CH 2 (Hf) porotons, which are close to the binding site of receptor L were affected and downfield shifted by the complexation of L with Cu 2+ . A detailed analysis of the 1 H NMR spectra reveals the significant changes of almost all the other proton signals in the ligand. For instance, the peak of aromatic ring hydrogen atoms (Hc, resonated at 7.35 ppm) was converted to two distinct peaks at 7.20 and 6.95 ppm (Hc 1 , Hc 2 ). In addition, signals belonging to the fluorene moieties and tert-butyl groups, which are quite far away from binding site, were also affected by complex formation. For example, the peaks of the tert-butyl groups of receptor L resonated at 1.29 (Hh 1 ) and 1.22 ppm (Hh 2 ) turned to one signal at 1.20 ppm during the titration.
All these observations might be attributed to the changes in electron density, the anisotropy effect and the changes in the conformation of receptor L during the complex formation. The fact that receptor L shows a sensitive recognition affinity to copper ions and a much more obvious shift of 1 H NMR peaks in the presence of Cu 2+ is actually not surprising because of a relatively rigid binding pocket of receptor L providing four sites of NH and OH which allows a suitable complex formation with a copper ion (Scheme 3).

Scheme 3: A proposed binding mode between L and Cu 2+ .
To show the high selectivity of chemosensor L for copper ions, the association constant and limit of detection (LOD) were compared with those reported for other related chemosensors (Table 1). It should be emphasized that due to different conditions applied to measure these values, we could not compare properly the performance of chemosensor L with those reported in literature. However, the associating constants of ligands having two moieties at the lower rim of a calix [4]arene scaffold were compared with the present chemosensor. The results indicated that the synthesized chemosensor L has a lower LOD and a higher K a in comparison with other chemosensors, which shows a stable and selective complexation with Cu 2+ .
benzothiazole [15] 18893 ± 1200 403 (ppb) 2-picolyl [36] 30221 ± 1600 196 ppb arylisoxazole [3] 1.58 × 10 4 benzimidazole [10] 7.24 × 10 9 96 ppb fluorene (this work) 1.8 × 10 6 96 ppb fluorescent probe for Cu 2+ detection. The sensitivity and selectivity of chemosensor L toward Cu 2+ has been investigated by spectroscopic methods. This sensor has a high affinity (K a = 1.8 × 10 6 M −1 ) for copper ions. The competitive metalion titrations by fluorescence spectroscopy showed that Cu 2+ can be selectively detected in the presence of other common metal ions. It is noteworthy to mention that the chemosensor L can be used as a "naked eye" indicator for Cu 2+ . Furthermore, fluorescence quenching of L upon addition of Cu 2+ showed a detection limit of about 9.6 × 10 −8 mol/L in the concentration range from 1 to 10 µM. This result demonstrated the possibility of quantitative detection of low levels of Cu 2+ in MeCN by using this chemosensor.

Experimental
Instruments and reagents 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker Avance III 400 MHz spectrometer. CDCl 3 and CD 3 CN were used as NMR solvents and TMS as internal standard. High-resolution mass spectra (HRMS) were recorded on a Qstar ESI-q-TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany). SPEKOL 2000 Analytik Jena spectrometer were used for recording of UV-vis spectra in acetonitrile as solvent. Fluorescence spectra were recorded on a JASCO FP-6500 spectrophotometer. All chemicals used in this paper including metal salts were purchased from Merck Company and were used without further purifications. Column chromatography was performed on silica gel (200-400 mesh). As starting material, p-tert-butylcalix [4]arene was synthesized according to the reported literature procedure [37].

2-Nitro-9H-fluorene (3) [39]
: 9H-Fluorene (6.0 g, 36.1 mmol) was dissolved in 100 mL of glacial acetic acid at 60 °C. 15 mL of nitric acid (65%) were added dropwise (~10 min) at 60 °C upon vigorous stirring. After the addition was completed, the resulting mixture was further stirred at 60 °C. The reaction was monitored by TLC (solvent EtOAc-n-heptane 1:9). After appearance of the spot of the dinitro product (≈100 min, R f ≈25%), the mixture was poured into 600 mL of water. The resulting crude product was filtered off, washed with water and recrystallized from 200 mL acetonitrile to give compound 3 (7.1 g, 92%) as slightly-yellow needles. 1 [4]arene (2, 0.50 g, 0.65 mmol) and N,N'-dicyclohexylcarbodiimide (2.6 mmol) was dissolved in 5 mL dichloromethane and stirred for 10 min at room temperature. 9H-Fluoren-2-amine (4, 0.20 g, 1.1 mmol) was added and the mixture was stirred for 24 h at room temperature. Then, the solvent was removed and the residue was dissolved in chloroform (50 mL) and washed with water (30 mL). The crude product was purified by silica gel column chromatography using petroleum ether- and L, HRMS of L, UV-vis and fluorescene titration spectra of L with Cu 2+ ion solutions.