A Novel Calix[4]Crown-Based 1,3,4-Oxadiazole as a Fluorescent Chemosensor for Copper(II) Ion Detection

The synthesis and characterization of a novel florescent chemosensor 1 with two different types of cationic binding sites have been reported in this work, which is a calix[4]crown derivative in 1,3-alternate conformation bearing two 2-phenyl-5-(4-dimethylaminopyenyl)-1,3,4-oxadiazole units. The recognition behaviors of 1 in dichloromethane/acetonitrile solution to alkali metal ions (Na+ and K+), alkaline earth metal ions (Mg2+ and Ca2+), and transition metal ions (Co2+, Ni2+, Zn2+, Cd2+, Cu2+, Mn2+, and Ag+) have been investigated by UV-Vis and fluorescence spectra. The fluorescence of 1 might be quenched selectively by Cu2+ due to the photo-induced electron transfer mechanism, and the quenched emission from 1 could be partly revived by the addition of Ca2+ or Mg2+; thus, the receptor 1 might be worked as an on–off switchable fluorescent chemosensor triggered by metal ion exchange.


INTRODUCTION
As the third most abundant transition metal ion after zinc and iron in the human body, copper is required by many living organisms for normal physiological processes (Turski and Thiele, 2009;Cotruvo Jr et al., 2015). Maintaining optimal concentration of Cu 2+ ion for living cells is an essential factor to keep the normal functioning of enzymes and intracellular metabolic balance. Thus, the development of new fluorescent chemosensors for Cu 2+ ion has drawn continuous interest during the past decades. The main progress in this area has been well reviewed (Cao et al., 2019;Sivaraman et al., 2018;Udhayakumari et al., 2017;Liu et al., 2017), and many fluorescent chemosensors for Cu 2+ ion based on various fluorophores such as coumarin (Zhang et al., 2019), Bodipy (Ömeroğlu et al., 2021), rhodamine (Fernandes and Raimundo, 2021), Schiff base (Singh et al., 2020), pyrene (Kowser et al., 2021), and 1,3,4-oxadiazole  have been reported by different research groups. Among these fluorescent chemosensors, the 1,3,4-oxadiazoles have drawn special interest due to their electron-deficient nature, high photoluminescence quantum yield, and excellent chemical stability, and have found practical applications in the fields of organic light-emitting diodes (Meng et al., 2020) and liquid crystals (Han et al., 2013;Han et al., 2015;Han et al., 2018). In addition, the nitrogen and oxygen atoms of the 1,3,4-oxadiazole unit can provide potential coordination sites with metal ions, which makes it usable as a signaling component in fluorescent chemosensors.
Calixarenes, as one kind of the most important super-molecules, have been widely used in design of fluorescent chemosensors for ions and neutral molecules due to their outstanding Edited by: Tony D. James, features such as preorganized binding sites, easy derivatization, and flexible three-dimensional structures (Kim et al., 2012;An et al., 2019;Miranda et al., 2019;Noruzi et al., 2019;Chen et al., 2020). Many calixarene-based fluorescent chemosensors for transition metal ions have been reported in recent years (Ma et al., 2015). However, the fluorescent switchable chemosensors triggered by different ions are quite few (Chung et al., 2007), which remains a challenge in the field of supramolecular chemistry. Herein, as part of our continuous research in the design and synthesis of new fluorescent chemosensors (Liu et al., 2021;Xie et al., 2016;Han et al., 2012), we utilize the 1,3-alternate calix[4]crown scaffold to construct an on-off switchable fluorescent chemosensor 1 in this work. The synthetic route for 1 is shown in Scheme 1. There are quite a number of chemosensors based on various macrocycles for copper detection reported in literatures (Lvova, et al., 2018;Doumani, et al., Kamei, et al., 2021), in which the macrocyles often only worked as receptors for Cu 2+ ions. In contrast, the chemosensor 1 in this work is special in that it has two kinds of macrocycles: one is from the 1,3-alternate calixarene, which provides a three-dimensional scaffold with two appending 1,3,4oxadiazole units as both signaling component and fluorophore; the other is from the calix[4]crown, which can bind the Mg 2+ or Ca 2+ ions and has an allosteric effect on the 1,3,4-oxadiazole units on opposite rings. The selective binding of 1,3,4oxadiazole with Cu 2+ ions results in the fluorescence quenching, while the binding of calix[4]crown with Mg 2+ or Ca 2+ ions can partly revive the fluorescence consequently. Thus, the compound 1 might work as a new type of switchable off-on fluorescent chemosensor.

Synthesis of 1
To a round-bottomed flask was added 6 (80 mg, 0.1 mmol), 10 ml of toluene, and 1 ml of thionyl chloride, and the mixture was refluxed for 5 h. After cooling, the solvent and the excess of thionyl chloride were removed at reduced pressure to give the benzoyl chloride, which was added to a solution of 4-(dimethylamino)benzohydrazide (39 mg, 0.22 mmol) in 10 ml of dichloromethane and 0.1 ml of pyridine. The reaction mixture was stirred for 12 h at ambient temperature and filtered. The precipitate was washed with ethanol to give the bishydrazide 7 as white solid, which was used to the next step reaction without further purification. The intermediate compound 7 was added to POCl 3 (5 ml), and the resultant solution was refluxed overnight under a nitrogen atmosphere. After the reaction mixture cooled to room temperature, it was poured into ice water and extracted with dichloromethane (3 × 10 ml). The combined organic layer was washed with water and brine, respectively. Then, the solvent was removed under reduced pressure, and the crude solid was purified by silica gel column chromatography using petroleum ether/ethyl acetate (1:1) 163.55, 158.76, 157.10, 152.20, 135.07, 133.83, 129.79, 128.32, 128.13, 122.75, 121.14, 118.35, 111.59, 111.27, 72.43, 72.24, 70.42, 70.14, 69.22, 40.11, 38.05, 22.43, 9.95 Figure S5, ESI).

General Procedures for the UV/Vis and Fluorescence Experiments
UV-vis spectra were recorded on a Cary 3,010 spectrophotometer, and the resolution was set at 1 nm. Steadystate emission spectra were recorded on a Varian Cary Eclipse spectrometer. For all measurements of fluorescence spectra, excitation was set at 334 nm for complexation, and the excitation and emission slit width was set to be 2.5 nm. Fluorescence titration experiments were performed with CH 2 Cl 2 solutions of compound 1 and varying concentrations of metal perchlorate in CH 3 CN solution. During all measurements, the temperature of the quartz sample cell and chamber was kept at 25°C.

Synthesis and Structural Analysis
As shown in Scheme 1, calix[4]arene 3 was reacted with tetraethylene glycol ditosylate in the presence of Cs 2 CO 3 to successfully afford the calix[4]crown 3 in 75% yield. The substitution reaction of 4 with CuCN gave 5 in 62% yield, which was refluxed with KOH in ethanol and treated with hydrochloric acid solution, readily providing the carboxylic acid 6 in good yield. Then, the carboxylic acid 6 was reacted with thionyl chloride, and treated with benzyol hydrazine or 4-N,N′dimethylaminobenzyol hydrazine to generate the intermediate bishydrazide 7, which was used in the next step without purification and refluxed with phosphorus oxychloride to afford the target products 1. Except for the calix[4]arene 3, all of the intermediate calix [4]crowns 3-6 and the chemosensor 1 are in 1,3-alternate conformation, which were well established by 1 H NMR and 13 C NMR data ( Supplementary Figures S1-S4, ESI). The 1,3-alternate conformation of 5 was further confirmed unambiguously by x-ray single crystal diffraction as shown in Figure 1. The x-ray crystallographic data are collected in Supplementary Table S1.

UV-Vis Absorption and Fluorescence Spectra Analysis
The selectivity of the receptor 1 toward different perchlorate salts, including Na + , K + , Mg 2+ , Ca 2+ , Co 2+ , Ni 2+ , Zn 2+ , Cd 2+ , Mn 2+ , Ag + , and Cu 2+ , was first investigated by UV-Vis spectroscopy. The UV-Vis absorption spectra for free 1 in CH 2 Cl 2 solution showed an intense and structureless absorption band (ε 4.94 × 10 5 L/mol·cm) peaking at 340 nm (Figure 2), which might have resulted from the spin-allowed π-π* transitions involving the phenyloxadiazole moiety (Han et al., 2006). The addition of Cu 2+ ions in the solution of 1 resulted in a significant decrease in the absorbance with an appreciable hypochromic shift of 20 nm. In contrast, only a slight decrease was observed upon addition of other metal ions mentioned above, which suggested that the selectivity of 1 toward Cu 2+ is much higher than the other metal ions.
Ion recognition ability of 1 was further studied by the fluorescence spectra. As shown in Figure 3, the receptor 1 exhibited a strong emission with λ max at 405 nm in solution of CH 2 Cl 2 . Upon addition of Na + , K + , and Mg 2+ , respectively, almost no changes were observed in the intensity and shape of the emission spectra of 1. It is noted that the addition of Ca 2+ might slightly increase the intensity with a bathochromic shift of ca. 15 nm, perhaps because the complexation between the Ca 2+ and the crown ether moiety changed the space distance of the two phenyloxadiazole units and the fluorescence changed consequently. Apparently, the fluorescence response of 1 toward transition metal ions was found to be more pronounced, and the addition of Co 2+ , Ni 2+ , Zn 2+ , and Ag + could quench the emission of 1 in a different extent, accompanied by a concomitant red shift of ca. 14-17 nm. In contrast, the addition of Cu 2+ significantly quenched the FIGURE 1 | X-ray molecular structure of 5.
Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 766442 fluorescence of 1 under the same conditions as the aforementioned metal ions, suggesting that there is a strong interaction between 1,3,4-oxadiazole moieties of 1 and Cu 2+ ion over the other metal ions.
The fluorescence emission properties of 1 in the presence of Cu 2+ and a competitive metal ion were measured to investigate the selective recognition for Cu 2+ . As shown in Figure 4, no apparent changes were observed in fluorescence intensity when 10 equivalent amounts of transition metal ions (Co 2+ , Ni 2+ , Zn 2+ , Cd 2+ , Mn 2+ , and Ag + ) were added to the solution of 1 and Cu 2+ (10 equiv). This suggested that the recognition for Cu 2+ was not interrupted by the competitive transition metal ions; thus, the receptor 1 might act as a selective fluorescent chemosensor for Cu 2+ . The addition of alkali metal ions (Na + and K + ) to the solution of 1 and Cu 2+ could increase the fluorescence intensity slightly, while the alkaline earth metal ions (Mg 2+ and Ca 2+ ) could revive the emission significantly.
The fluorescence changes of 1 upon addition of Cu 2+ and Mg 2+ ions are displayed in Figure 6. The nitrogen atoms of the 1,3,4-oxadiazle units can bind with Cu 2+ to form the complex 1·Cu 2+ , and the paramagnetic nature of Cu 2+ ion could strongly quench the fluorescence of the 1,3,4-oxadiazole units through the electron transfer mechanism, which is consistent to the results reported in literature (Han et al., 2012). In contrast, the polyether ring (crown-5 moiety) and the oxygens from the two propoxyl groups could provide coordination sites with the alkaline earth metal ions to form the complex 1·Mg 2+ , which will change the molecular conformation as well as the space distance of the two 1,3,4-oxadiazole units. Consequently, the decomplexations between the 1,3,4-oxadiazoles and Cu 2+ ions took place and resulted in the increase of the fluorescence. Thus, the receptor 1 might be acted as an on-off-on switchable fluorescent chemosensor triggered by the exchange of Cu 2+ and Mg 2+ .
FIGURE 4 | Fluorescence spectra (λ exc 334 nm, Slit 2.5) of 1 (1 × 10 −6 mol/L) and Cu 2+ (10 equiv) upon addition of other metal ions (10 equiv) in CH 2 Cl 2 /CH 3 CN (1,000:1, v/v). To gain a better understanding about the switchable fluorescence of the chemosensor 1, DFT calculations with the GAUSSIAN 09 series of programs (Frisch et al., 2013) were carried out to analyze the molecular structures of 1 and 1·Mg 2+ , and DFT method B3-LYP with 6-31G(d) basis set was used for geometry optimizations (A. D. Becke, 1993). As shown in Figure 7, the distance between N1 and N2 in the free receptor 1 is 9.97 Å, while the corresponding distance is 11.74 Å in the complex 1·Mg 2+ , indicating that the molecular conformation changed simultaneously due to the allosteric effect (Kumar et al., 2012;Ni et al., 2013). The conformational change as well as the increase in distance makes it difficult for the chemosensor 1 to coordinate with Cu 2+ ion to form the stable complex, which reasonably explains the fact that the addition of Mg 2+ ions to the solution of 1 and Cu 2+ can trigger the revival of fluorescence.

CONCLUSION
In summary, we have designed a new type of fluorescent chemosensor based on a 1,3-alternate calix[4]crown with two different cationic binding sites. The 1,3,4-oxadiazole units could bind selectively with Cu 2+ to form the complexation and resulted in the fluorescence quenching of the chemosensor. The presence of various transition metal ions does not interfere with the quenching process, while the alkaline earth metal ions Mg 2+ might be entrapped by the crown-5 moiety and revive the fluorescence significantly due to the allosteric effect. As the  Frontiers in Chemistry | www.frontiersin.org November 2021 | Volume 9 | Article 766442 6 chemosensor in this work is not soluble in water, it is difficult to investigate the Cu 2+ ions' detection under physiological conditions. Devising a water-soluble chemosensor for Cu 2+ ions is in progress in our lab.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.