Fluorescent Calix[4]arene-Oxacyclophane Sensor for Transition Metal Cations

: The sensing behavior of a calix[4]arene-oxacyclophane-carbazole conjugate ( 1 ) towards Cu(I) ions was investigated. Contrary to the noteworthy sensitivity and selectivity previously found for Cu(II), the affinity of 1 for Cu(I) cations is much lower. Through fluorescence titration assays, the apparent binding constants ( K a ) for the two ions were determined, showing that an affinity of over thirteen fold was displayed for Cu(II). Two Cu(I) counter-ions were used to determine possible effects on the binding event. It was found that acetate and iodide ions behave similarly, yielding K a of the same magnitude. Formation of a ground-state supramolecular complex between 1 and Cu(I) ions was not observed on UV-Vis titrations, in contrast to what was previously reported for Cu(II) using the same host. The affinity of 1 for Fe(III), a major biological competitor, was also assessed. The fluorescence of host 1 is quenched by Fe(III) ions although to a lesser extent (32% less efficient than Cu(II)). The study demonstrates that calixarene 1 is able to differentiate copper ions in two common oxidation states using fluorescence techniques, thereby suggesting its application for redox-active centers in biomimetic chemistry.


Introduction
Development of sensitive and selective chemosensors for trace detection of transition metal cations (e.g., copper(II), copper(I) and iron(III) ions) abundant in nature, either associated with important biological processes or representing environmental hazards and health issues, is an attractive area of recent research [1][2][3]. Copper and iron cations are involved in crucial metabolic processes in the human body, such as cellular respiration, muscle contraction, enzyme synthesis and regulation of acid-base balance and osmotic pressure in cells [4,5].
Calix [4] arenes have been studied extensively as one of the most widespread scaffolds in host-guest chemistry because of their rigid structures (particularly important when the recognition and reporting events are to be undertaken in the fluid phase), making them perfect candidates for complexation studies with ions and neutral molecules [6,7]. Fluorescent calixarene-containing receptors have proven applications as sensors for a wide range of metal ions [7].
Calix [4]arene-oxacyclophane molecular receptors, either standing alone or integrated in a conjugated polymer chain, were recently described by us as excellent platforms for the recognition of Cu(II), with an outstanding selectivity by comparison with other tested metal ions, (Hg(II) and Pb(II)) [8]. As a continuation of our previous  studies, we were interested in determining the response of the same calixarene receptor 1 towards Cu(I), the ubiquitous counterpart of Cu(II) in biological redox activities. In addition, Fe(III), a d-block competitor ion in biological media, was also used in the study. This communication reports the preliminary results regarding the binding affinities of bicyclic calix [4]arene-carbazole probe 1 to Cu(I) and Fe(III) in organic media.

Instruments and Methods
Steady-state fluorescence spectra were acquired on a Perkin Elmer LS45 fluorimeter using a 1-cm quartz cuvette in right angle (RA) geometry at 25 °C in air-equilibrated conditions.
The titration studies were carried out at a constant concentration of the probe (1.0 × 10 −7 M) in a mixture of CH3CN:CHCl3 (1:1) using known amounts of metal cations in fluorophore solutions at 25 °C, being the spectra acquired after each addition. Copper acetate was previously solubilized in pyridine and added to the probe solution in CH3CN:CHCl3 (1:1).

Sensing of Metal Cations
Since a remarkable affinity of Calix-OCP-2-CBZ (compound 1; Chart 1) towards Cu(II) in organic media has been disclosed [8], further studies were foreseen to explore the behavior of such sensors to other biologically important transition metal ions, in particular copper (I) owing to its significance in biological systems where it appears in Cu(I)/Cu(II) redox pairs. In this report, the preliminary results on the recognition abilities of 1 to Cu(I) are described. The sensitivity of receptor 1 towards this cation, as copper(I) acetate and copper (I) iodide, was evaluated through fluorimetric titrations using diluted CH3CN:CHCl3 (1:1) solutions of fluorescent sensor 1 (1 × 10 −7 M). Due to the limited solubility of CuOAc in the solvent mixture, the salt was previously solubilized in pyridine and then added to the solution of the fluorophore for titration assays. The photostability of 1 in the solvent mixture was first evaluated under the conditions used in the titration experiments (λexc = 380 nm, RA illumination). Fluorescence emission data have shown that no photodegradation occurred up to 1 h of continuous irradiation. It was also verified that, in the range of concentrations used, no inner-filter effects ascribable to the absorption of incident radiation at the excitation (380 nm) or emission radiation from the fluorophore (390-550 nm) by the metal cations were present, at least to an extent that would require a proper mathematical correction.
As depicted in Figure 1a,b, the emission of calixarene 1 in the presence of Cu(I), whatever the counter-ion used, is extremely reduced. Indeed, the emission of 1 is only quenched by 7.9% after addition of 300 equiv. of copper acetate salt. When the iodide salt was used, a similar response (6.6% attenuation of emission after addition of 300 equiv. of the salt) was found. This outcome discards the possible influence of significant effects of the anions on the binding strengths attained in the complexes. Fe(III), the competitor ion chosen for the study, decreases the initial fluorescence intensity of 1 by 31% upon contact with 300 molar equivalents of this cation. The spectra obtained for this titration are shown in Figure 1c. For comparison purposes, the spectra obtained from the titration experiment with Cu(II), as its perchlorate salt, are also shown in Figure 1d. In this case, the quenching efficiency reaches 84% upon addition of 300 equiv. of the salt.
The binding constants for the three salts were roughly estimated by the Stern-Volmer formalism, assuming a 1:1 stoichiometry for all the intervening complexes. A binding constant (or association constant (Ka)) of 2.87 × 10 3 M −1 was retrieved for Cu(I), while for Fe(III) and Cu(II) the values were Ka = 1.25 × 10 4 M −1 and Ka = 3.85 × 10 4 M −1 , respectively.
Evidence for the formation of ground-state complexes between 1 and the metal cations Cu(I) and Fe(III) was investigated by UV-Vis titrations. Contrary to what was previously observed for Cu(II) [8], no evidence for complexation was found, as judged by the absence of any isosbestic points in the spectra (Figure 2a,b). Thus, even for Fe(III), which showed a higher affinity to calixarene 1 compared to Cu(I), the equilibrium concentration of the charge-transfer complex should be relatively low to be observed by UV-Vis spectroscopy.
Solely based on one of our previous observations [8] that the relative success of the binding event will rely, in first place, on the match between the size of the ligand cavity in calixarene 1 and the size of the incoming cation, one would expect a much larger affinity of 1 to Cu(I) since its ionic radius is very close (rion = 0.60 Å) to that of Cu(II) (rion = 0.57 Å), both in a square planar tetra-coordinate geometry [10]. Of course, besides that parameter, the scant magnitude of the charge-transfer interactions between 1 and Cu(I) seems to dominate the observed outcome in this case. Further studies are needed to shed light on this issue. The same observation can be made for Fe(III), which has an ionic radius of 0.49 Å (square planar configuration) [10].

Conclusions
The above-mentioned results indicate that Calix-OCP-2-CBZ (1) is poorly responsive to the presence of copper ions in its reduced oxidation state in an organic solvent matrix. This can be considered an excellent observation since it prospectively allows sensor 1 to selectively discriminate between the two most prevalent forms of copper ions in biological processes, namely those occurring in redox-active centers. The binding affinity of 1 for Fe(III) was also explored and a lower sensitivity was obtained when compared to Cu(II).
Deeper insights on the recognition events underpinning the reported behaviors here described, and those of other relevant transition metal cations, are currently being investigated and will be reported in due course.