Extraction and complexation of alkali and alkaline earth metal cations by lower-rim calix [ 4 ] arene diethylene glycol amide derivatives †

Novel calix[4]arene derivatives, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetrakis(N-(2-(2-methoxyethoxy)ethyl)carbamoyl methoxy)calix[4]arene (1) and 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra(N,N-bis(2-(2methoxyethoxy)ethyl)carbamoyl methoxy)calix[4]arene (2), were prepared by introducing diethylene glycol subunits at the lower calixarene rim. The complexation affinities of these compounds towards alkali and alkaline earth metal cations were studied at 25 1C in acetonitrile and methanol by means of spectrophotometric, conductometric, and potentiometric titrations. The stability constants of the corresponding complexes with 1 : 1 (cation : ligand) stoichiometry were determined (in some cases only estimated), and their values obtained by different methods were in good agreement. The complexes with secondary-amide derivative (1) were observed to have much lower stabilities than those with tertiary-amide derivative (2). This was presumably mostly caused by the presence of intramolecular NH OQC hydrogen bonds in the former case. It was found that solvent significantly affected the stability of the complexes; the prepared calixarenes showed considerably higher affinities for cations in acetonitrile than in methanol. Generally, the ligands studied showed better binding abilities for alkaline earth cations than for alkali metal cations. The extraction of metal picrates from water to dichloromethane by the complexation of metal ions with ligands 1 and 2 was also investigated. In accordance with the complex stabilities, all cations were extracted fairly well (in some cases even excellently) with the tertiary-amide derivative, whereas this was not the case in the extraction experiments with the other ligand studied.


Introduction
Complexation properties of calixarene derivatives have been extensively studied due to their ability to selectively bind a wide variety of guest species. [1][2][3] Numerous macrocyclic receptors have been prepared by functionalization of calixarenes at the lower and/or upper rim. By choosing adequate number of phenolic units and appropriate substituents, it has been possible to prepare selective and efficient calixarene hosts for cations, 4,5 , anions, 4,6,7 and neutral species.

4,8-10
The p-tert-butylcalix [4]arene derivatives containing carbonyl groups at the lower rim (ketones, esters, amides) were reported to possess excellent properties for binding alkali, alkaline earth, and transition metal cations. 5,11 Secondary- [11][12][13] and tertiary-amide [14][15][16] calixarene derivatives were shown to have particularly high affinity towards alkali and alkaline earth metal cations. Formation of intramolecular NH•••O=C hydrogen bonds in tetrasubstituted secondary-amide derivatives was proven to have strong influence on the binding properties of such ligands. 12,13,[17][18][19][20][21][22][23][24] In addition to the above mentioned calixarene size and binding groups nature, its affinity towards metal cations depends strongly on the reaction medium, i.e. on the solvent used. [11][12][13][14]17,[25][26][27][28][29][30][31] Several ethylene glycol-based calixarene derivatives have been reported previously. 1,[32][33][34][35][36][37][38][39] The first oxyalkylated p-tertbutylphenol-formaldehyde oligomers (both linear and cyclic) were developed as demulsifiers for petroleum industry. 1 Calixarene crown ethers with ethylene glycol substituents at the lower rim were shown to selectively bind sodium, potassium, and cesium cations. 32,33 Another interesting group of compounds are bis-calixarenes, also known as calix-tubes, which possess two calixarene skeletons bridged by ethylene glycol or various polyethylene glycol (PGE) chains. 34 Calixarene derivatives with a large number of attached (poly)ethylene glycol groups have an increased hydrophilic character, and have found application as agents for extraction of metal cations from aqueous to organic phase. [35][36][37] In order to optimize the structure of calix [4]arene derivatives for metalion biphasic extraction and phase-transition catalysis, Shinkai et al. 35 prepared several amphyphilic calix [4]arene derivatives by introducing hydrophobic groups at the upper calixarene rim and oligo(ethylene glycol) chains at the lower one. In the course of an extraction process, the latter hydrophilic groups penetrate to the aqueous layer, whereas the hydrophobic calixarene basket remains in a non-polar organic layer. In that way such compounds can efficiently bind cations in aqueous phase and transfer them to the organic one. Interestingly, the length of the hydrophilic chains does not significantly affect the ligand extraction and phase-transfer catalysis properties.
On the other hand, the receptor can be improved in these respects by increasing its overall lipophilicity, which can be accomplished by appending the larger lipophilic functionalities to the upper calixarene rim. 34 Roundhill et al. 36 synthesized a number of polyethylene glycol functionalized calix [4]arenes by introducing PEG groups at both calixarene rims, and the obtained compounds were envisaged as potential efficient metal-ion host molecules and extracting agents. A larger ethylene glycol-based calix [6]arene derivative 37 was investigated as catalyst and extraction agent for alkali metal cations, whereby the solvent effect on the ligand abilities was proven to be of great importance. Shi and Zhang 38 reported a water-soluble p-tert-butycalix [8]arene bearing PGE chains which was capable of efficiently binding organic molecules and ions in its hydrophobic cavity. Most recently, pegylated octopus-shaped calix [4]arenes with different degree of polyoxyethylation of the lower-rim substituents were described as promising supramolecular drug delivery platforms. 39,40 To the best of our knowledge, there is only one calix [4]arene amide derivative bearing diethylene glycol chains bound to the amide nitrogen atoms reported in the literature, 41 which was studied as anion 41 and amino acids [42][43][44] receptor.
In this work, we present the syntheses of calix [4]arene amide derivatives with diethylene glycol functionalities appended to amide group at the lower rim, and the study of their complexation affinities towards alkali and alkaline earth metal cations in two solvents with different solvation and hydrogen-bonding abilities (methanol, MeOH, and acetonitrile, MeCN). In addition, the efficiencies of the extraction of cations from water to chloroform with both ligands has been investigated and discussed.

Syntheses
The syntheses of diethylene glycol calixarenes 1 and 2 were performed in several reaction steps, as shown in Scheme 1. Compound 1 was prepared via aminolysis (i) of the p-tertbutylcalix [4]arene tetraethyl ester with 2-(2-methoxyethoxy) ethanamine with 90 % yield, as described in the Experimental section. Compound 2 was synthesized by the modified method described previously, using tetra acid chloride p-tertbutylcalix [4]arene, 45 starting from the corresponding tetraester (cone conformation) by (ii) hydrolysis to the tetraacid, (iii) activation to the acid chloride, and (iv) amide bond formation (65 % yield). For both compounds an additional purification step was required to ensure that all of the ions were removed, and this was done in a similar manner as described in ref. 46. Details of purification procedure are described in the Experimental section. Scheme 1 Syntheses of compounds 1 and 2. Reagents and conditions: (i) tertbutanol, r.t.; (ii); a) NaOH, EtOH/H2O, Calixarene derivatives 1 and 2 were characterized by spectroscopic methods and mass spectrometry. The 1 H NMR spectra of compounds 1 and 2 (CDCl 3 ) showed the pattern characteristic of p-tert-butyl-calix [4]arene in a cone conformation and approximately C 4 symmetry of tetrasubstituted calix [4]arene. 19 Two singlets appeared, one corresponding to tert-butyl groups (1.10 ppm, 1.09 ppm) and another due to the calixarene aromatic protons (6.79 ppm, 6.77 ppm). In addition, two doublets assigned to the equatorial (3.16 ppm, 3.26 ppm) and axial (4.65 ppm; 5.07 ppm) bridging methylene protons could be found. In the spectrum of compound 1, a rather high chemical shift of the amide protons (7.94 ppm) indicated the presence of intramolecular NH•••O=C hydrogen bonds between the amide groups of the lower-rim substituents. 19,20 The FTIR data were fully in agreement with NMR results. In the spectrum of 1 the NH stretching band at 3373 cm -1 corresponded to intramolecular hydrogen-bonds between the amide groups. Positive ESI mass spectra of compounds 1 and 2 were acquired in acetonitrile. proposed fragmentation pathways of 1 and 2 are shown in Schemes S1 and S2 (ESI).

Cation complexation studies
The hypochromic effect on the UV spectra of the acetonitrile solutions of 1 and 2 was observed upon stepwise addition of LiClO 4 , NaClO 4 , KClO 4 , RbNO 3 , CsNO 3 , Mg(ClO 4 ) 2 , Ca(ClO 4 ) 2 , Sr(ClO 4 ) 2 , and Ba(ClO 4 ) 2 (Figs. S5-S18, ESI) solutions. In addition, an isosbestic point at 256 nm appeared in the case of ligand 1 titrations with Mg(ClO 4 ) 2 (Fig. S12, ESI) and Ba(ClO 4 ) 2 ( Fig. 1). Isosbestic points were also observed in ligand 2 titrations with Rb + (251 nm), Cs + (254 nm), and Ba 2+ (255 nm) (Figs. S10, S11, and S18, ESI). Linear dependence of absorbance vs. the amount of cation added up to the ratio n(cation)/n(ligand) ≈ 1, followed by the break in the titration curve, indicated a strong complexation and formation of 1:1 complexes ( Fig. 1; the corresponding stability constants could only be estimated, Table 1). This was observed in all above mentioned titrations, except in that of 1 with KClO 4 and RbNO 3 , and those of both 1 and 2 with CsNO 3 . In the case of complexes K1 + and Cs2 + , their stability constants could be determined spectrophotometrically (Fig. 2, Fig. S11, ESI, Table 1). Addition of RbNO 3 and CsNO 3 into the calixarene derivative 1 acetonitrile solution had no significant effect on its UV spectrum, indicating that under the conditions used no observable complexation took place. To corroborate the findings obtained by spectrophotometry, conductometric titrations of acetonitrile solutions of alkali and alkaline earth cation salts with calixarene derivatives 1 and 2 were carried out (Figs. S19-S33, ESI). In most cases a linear decrease in molar conductivities with the addition of calixarene solutions was recorded up to a break in the titration curve at the molar ratio n(ligand)/n(cation) ≈ 1, indicating, as for spectrophotometric titrations, a strong complexation and formation of 1:1 complexes (example can be seen in Fig. 3). The exceptions were titrations of KClO 4 with 1 ( Fig. 4) and CsNO 3 with 2 ( Fig.  S25, ESI). By processing the data of these titrations, the stability constants of K1 + and Cs2 + were determined. In all the above cases, decrease in the molar conductivity was due to the lower electric mobility of the larger complexes compared to the free metal cations. Like with spectrophotometric experiments, during the conductometric titrations of RbNO 3 and CsNO 3 acetonitrile solutions with ligand 1 no complexation was observed under the experimental titration conditions. The stability constants of the Na1 + and Na2 + complexes in acetonitrile were too high for spectrophotometric or conductometric determination. For that reason, direct potentiometry using a sodium-selective glass electrode was applied. Potentiometric titration curves for both ligands showed a steep p[Na] jump at the 1:1 n(ligand)/n(cation) ratio, which is in accordance with the results of previously mentioned methods. However, the p[Na] jump was steeper in the case of titration with tertiary-amide derivative 2, indicating a higher stability of the corresponding complex. The jump was even too steep (Fig. S34, ESI) to allow accurate calculation of the Na2 + complex stability constant so it could only be estimated. On the other hand, stability constant of Na1 + complex was determined by processing the corresponding potentiometric titration data (Fig. 5, Table 1).    6). The stability constants of these complexes are given in complex was observed conductometrically, although no complexation was detected using spectrophotometry. The stability constant of the Na2 + complex in methanol was, like in acetonitrile, too high for spectrophotometric and conductometric determinations. For that reason, it was determined by means of direct potentiometric titration (Fig. 7, Table 1). As stability of the sodium complex with 1 was much lower (Table 1), to determine its stability constant potentiometrically considerably higher concentrations of both ligand and sodium perchlorate were needed. Because of the limited solubility of calixarene 1 in methanol these measurements were not conducted. Given the data listed in Table 1, it is evident that the affinity of ligand 2 for alkali and alkaline earth metal cations is much higher than that of ligand 1. The main reason for the difference can be readily explained by taking into account the presence of N-H•••O=C intramolecular hydrogen bonds in the latter case. Namely, in order for a cation to form a complex with 1, these bonds need to be disrupted to allow reorganization of the amide groups. Amide hydrogen atoms are in fact competing with the cation for the carbonyl oxygen. Tertiary amide derivatives, like 2, do not have the ability to form the aforementioned hydrogen bonds, so no unfavorable competition between the cation and -NH group could take place. Secondary-amide derivative 1 binds smaller Li + and Na + cations very well. Its affinity for larger K + is lower, whereas Rb + and Cs + are too large to fit into its hydrophilic cavity. On the other hand, this compound binds all alkaline earth metal cations quite strongly in acetonitrile, as expected due to their size and charge. Tertiary-amide derivative 2 forms highly stable complexes with all cations, except with the largest Cs + (even in that case the complex stability is moderate). This sizeand charge-dependent selectivity is even more pronounced in methanol. As in MeCN, in MeOH both ligands 1 and 2 showed higher affinity towards alkaline earth compared to alkali metal cations (the only exception is complexation with Ba 2+ because of its relatively large size). However, the complex stabilities in methanol are considerably lower ( . Ligand solvation effect on the studied equilibria should be presumably more pronounced in reactions with 1 than in those with 2. In both cases methanol molecules compete with the cation for binding sites by forming hydrogen bonds with amide carbonyl oxygen atoms. However, contrary to 2, in the case of 1, which is secondary amide derivative, MeCN molecules can (as a proton acceptor) form H-bonds with amide NH groups of the receptor. This in turn leads to disruption of intramolecular N-H•••O=C hydrogen bonds and to orientation of carbonyl groups favorable for the complexation of metal ion. Obviously the solvation of the complex also plays an important role in determining its thermodynamic stability. However, on the basis of data presented in this paper we cannot say much about this effect.
It should be also mentioned that the process of solvent molecule inclusion into the hydrophobic cavities of calixarenes 1 and 2 and their complexes could occur. This phenomenon is more pronounced in acetonitrile than in methanol, and could be quite important in determining the equilibria of the complexation reactions. 12,13,28,48-50 Table 1 Stability constants for alkali and alkaline earth metal complexes of ligands 1 and 2) in acetonitrile and methanol at 25.0 o C. Uncertainties are given in parentheses as standard errors of the mean (N = 3 to 5).

Extraction properties
The abilities of compounds 1 and 2 to extract alkali and alkaline earth metal cations from water to dichloromethane were explored according to the procedure described in detail in the Experimental section. As can be seen by inspecting the data in Table 2, secondary-amide derivative 1 moderately extracted sodium, potassium, and calcium picrates from aqueous to organic phase. Other examined picrate salts were extracted poorly (or not at all) by this compound. On the other hand, tertiary-amide derivative 2 was shown to be much better extractant of alkali and alkaline earth metal cations. Most of them were extracted very well (95-100 %) by 2, the exception being rubidium, cesium, and magnesium ions which were not extracted as efficiently. It is interesting to note that extraction efficiency was 100 % in the case of sodium cation, which was not observed earlier with the other calix [4]arene tertiary-amide derivatives. 16,45 The results described are obviously directly correlated with the stabilities of the corresponding complexes, i.e. by the structural features of the compounds studied, and their amphyphilic character.

Conclusion
The diethylene glycol calix [4]arene derivatives 1 and 2 were designed and synthesised as compounds comprising hydrophobic and relatively hydrophilic parts as well as binding site which ensure strong complexation of alkali and alkaline earth metal cations. Hence, due to these properties their strong cation binding associated with high extraction abilities was expected. The complexation of alkali and alkaline earth cations by these ligands was studied by several experimental methods. The solvent effect on the complexation reactions was also examined using two solvents with different solvation and hydrogen-bonding abilities, namely acetonitrile and methanol. In both solvents the affinity for binding alkali and alkaline earth metal cations was found to be much higher in the case of tertiary-amide derivative 2 as compared to compound 1 which comprises secondary-amide subunits. That can be explained mostly by taking into account the presence of intramolecular NH•••O=C hydrogen bonds in 1, which cannot be formed in 2. In the process of cation complexation, these bonds have to be disrupted, which is energetically quite demanding and therefore significantly reduces the complex stability.
The hydrogen-bonding role is also important for the explanation of the solvent effect on the equilibria of the complexation reactions. Namely, carbonyl-oxygen binding sites of compound 1 are most probably "blocked" by the formation of H-bonds with methanol molecules as a proton donors, whereas such bonds cannot be established with acetonitrile. By considering also the difference in cations solvation in the two solvents, the much higher complex stabilities in MeCN in comparison to MeOH becomes obvious.
In accordance with the above mentioned results, the structural differences between compounds 1 and 2 were shown to be of utmost importance in determining their abilities for extraction of metal cations from water to dichloromethane. Thus, contrary to 1, calix [4]arene derivative 2 was found to be a very good, even excellent extractant of alkali and alkaline earth metal picrates, with 100 % efficiency in the case of sodium cation. Therefore, it can be concluded that, due to its amphyphilic character and strong cationbinding ability, compound 2 can be considered as a promising reagent for the extraction of metal ions from aqueous to organic phase.

Experimental General
All reagents used in the syntheses were of the best grade commercially available and were not further purified. Solvents were purified by standard procedures. 51 Analytical TLC was performed on silica gel plates (SiO 2 , Merck 60 F254). Melting points were determined on Kofler hot-bench apparatus and were not corrected. 1  Compounds p-tert-calix [4]arene ethyl ester, 52 tetra acid chloride of p-tert-calix [4]arene, 15 2-(2-metoxyetoxy)ethyl amine, 53 and bis(2-(2-methoxyethoxy)ethyl) amine 54 used in the synthesis of 1 and 2 (Scheme 1) were prepared according to the procedures described in the literature.  conductivities were corrected for the conductivity of the solvent. Titrations for each system were done in triplicate. The obtained data were processed by the OriginPro 7.5 program.

Potentiometry
The stability constants of Na1 + and Na2