Laurent Bouteiller

Benjamin Isare

Guylaine Ducouret

François Lequeux

Tuning reversible supramolecular polymer properties through co-monomer addition

Keywords: supramolecular polymer, organogelator, hydrogen bond, bis-urea supramolecular polymer, organogelator, hydrogen bond, bis-urea

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Tuning reversible supramolecular polymer properties through co-monomer addition

Introduction

A large variety of low molar mass compounds are known to form thermoreversible gels at low concentrations in organic solvents. 1 The overwhelming majority of these organogels are obtained by cooling a hot solution of the organogelator below a critical temperature where molecular aggregation or even crystallization occurs. If the crystals obtained are highly anisotropic, they can form a percolating network, which effectively entraps the solvent. These classical organogels are intrinsically metastable, because close packing of the crystalline fibres, which is a thermodynamically favourable process, leads to a macroscopically biphasic system. However, in some cases this process is kinetically trapped, and the gels obtained are effectively stable for months and can be used in various applications. 1 In contrast to these materials, a few hydrogen bonded low molar mass compounds have been reported to form thermodynamically stable viscoelastic solutions. [START_REF] Hanabusa | [END_REF][3][4][5] Rather than thick crystalline fibres, these reversible polymers 6 form filaments with a cross-section close to the molecular dimension of the monomer, so that a dynamic exchange between the filaments is possible at room temperature. If the filaments are sufficiently rigid, they can entangle at low concentrations and yield elastic properties at high frequencies. On the other hand, the dynamic nature of the filaments is responsible for viscous flow at low frequencies, because of the reversible breaking of the filaments. The dynamic nature of the filaments is also responsible for the thermodynamic stability, for the self-healing behaviour, and even for the easily tunable properties of the viscoelastic solutions. Indeed, for a thermodynamically stable system, a small incremental change can be expected to lead to a small property variation. This is in contrast to classical organogels, which can be totally disrupted even by a small incremental change, because of their metastable condition. The purpose of this article is to show that tuning the properties of rigid reversible supramolecular polymers by adding co-monomers is indeed a straightforward and efficient methodology.

Results and discussion

Reversible homopolymer structure and properties

Three bis-urea based monomers (Fig. 1) were considered for this study. The self-assembly of monomer EHUT has been previously characterized by FTIR spectroscopy, small angle neutron scattering (SANS) and rheology. [8][9][10] At room temperature, in toluene or dodecane solutions, EHUT forms thick and probably tubular filaments with about three molecules in the cross-section. This supramolecular structure is responsible for the strong viscoelasticity of the solutions (see below). The three monomers considered here (Fig. 1) contain the same associating bis-urea unit and thus behave similarly: SANS analyses have shown that all three monomers self-assemble to form thick filaments with the same diameter and the same number of molecules in the cross-section (Table 1). 8 At high temperatures or at low concentrations, the thick structure disappears in favour of thinner filaments containing a single molecule in the cross-section. 8,11 The thin filaments do not show viscoelasticity, due to faster dynamics and reduced rigidity of the filaments. The thermal stability of the thick filaments has been tested by FTIR spectroscopy. 8 The values of the transition temperature above which the thick filaments are unstable (T**) are mentioned in Table 1: the thermal stability of the thick filaments increases in the order BuPEH < EHUT < DMHUT. The stability of the thick filaments to dilution can be conveniently tested by Isothermal Titration Calorimetry (ITC). 12 The principle of the technique is to measure the heat evolved while aliquots of a relatively concentrated bis-urea solution are injected into pure solvent (placed in a calorimetric cell, at a fixed temperature). Fig. 2a shows the heat-flow curves obtained when a 0.24mM bis-urea solution in toluene is incrementally injected into pure toluene at 25 °C, and Fig. 2b shows the enthalpograms deduced from the heat-flow curves after integration. The three bis-ureas show qualitatively the same behaviour, but some significant differences are visible. First, the dissociation of the thick filaments is an endothermal process for all three bis-ureas, but Fig. 2a shows that the dissociation of the thick filaments is a slower process for DMHUT than for the other bis-ureas. Second, each enthalpogram (Fig. 2b) displays two thresholds: 8 the low concentration threshold corresponds to the transition between the thick filaments and the monomers and is not of interest for the present discussion. The second threshold corresponds to the transition between the thick filaments and the thin filaments: its position gives the critical concentration (C**) below which the thick filaments are unstable.# The values of C** are mentioned in Table 1, and they show that the stability toward dilution follows the same trend as the thermal stability: the thick filaments stability increases in the order BuPEH < EHUT < DMHUT.

Simple visual inspection reveals that all three monomers form gels in toluene. Consequently, rheology was used to see if the quantitative differences in the strength of the self-assembly have measurable consequences on the material properties. The experiments were performed in dodecane to avoid artefacts due to solvent evaporation. The stability of the thick filaments is enhanced in dodecane, compared to toluene, but no precise values fot T** and C** could be obtained. Fig. 3 shows the frequency dependence of the storage and loss moduli for EHUT and DMHUT solutions. ‡ The results for EHUT (Fig. 3a) prove the viscoelastic nature of the solution. At low frequencies, the mechanical response is dominated by the loss modulus. At higher frequencies the storage modulus is predominant and reaches a plateau value. This signature has previously been interpreted to be due to the entanglement of long and semi-flexible filaments presenting a fast scission and recombination process. 5,10 DMHUT also shows this characteristic signature, but only at higher temperature (Fig. 3c).

Comparison of data at the same temperature and concentration (Fig. 3a and3b) shows that the 3 characteristic relaxation time (the crossing point of G' and G") for the DMHUT solution is at least two orders of magnitude smaller than in the case of the EHUT solution. This means that the filaments present in the DMHUT solution have slower dynamics. This effect is probably related to the slow dissociation revealed by ITC (Fig. 2a).

Influence of co-monomer composition on transition temperature

Because of the difference in filament stability between the three monomers, it is of interest to mix them and see if tuning of the properties is possible. Figure 4 shows the evolution of the ratio of FTIR absorbances for EHUT/DMHUT mixtures in toluene. This ratio measures the shape of the vibration band for hydrogen bonded N-H groups and is a characteristic of the filament structure: a high value (c.a. 1.25) is attributed to the thick filament structure, whereas a low value (c.a. 1.1) is attributed to the thin filament structure. 8 Figure 4 shows that the monomer mixtures behave similarly to the pure components, but with a gradual shift of the transition temperature. This means first that copolymers are indeed obtained: if a noninteracting mixture of EHUT and DMHUT filaments were obtained, then two transitions should be detected. A second point worth noting is that the slope of the curves (that is, the cooperativity of the transition) is the same in the case of the copolymers as in the case of the homopolymers. This observation may seem surprising, because based on the monomer structures, statistical copolymers are expected, which means that a broadening of the transition could be expected because of compositional heterogeneity within a copolymer sample. Indeed, in a given mixture, the filaments with a content in EHUT higher than average should present a transition temperature at a lower temperature than the average. However, due to the great length of the filaments, the probability to find a pure EHUT filament in an equimolar EHUT/DMHUT solution is vanishingly small. Consequently, the sharpness of the transition for a mixture means that all the filaments in the sample have a very similar overall composition (the average composition). Compositional fluctuations along the filament most probably occur, but do not affect the slope of the transition because the fluctuations are averaged out over the very long filaments.

Finally, figure 5 shows that, for both monomer mixtures EHUT/DMHUT and EHUT/BuPEH, the transition temperature is close to a linear function of the composition. From a practical point of view, this point is particularly useful because it means that the temperature above which the viscoelastic properties of the solution disappear, can be finely tuned.

Influence of co-monomer composition on dilute solution viscosity

In order to check if the co-monomer composition also has an influence on properties far from the transition temperature, dilute solution viscosity measurements were performed at 25°C. Figure 6 shows that the viscosity of the DMHUT solution is much larger than for EHUT: the increased stability of the DMHUT filaments probably means that the DMHUT filaments are longer than the EHUT filaments, explaining the larger viscosity. As a consequence, the viscosity of EHUT/DMHUT mixtures is strongly influenced by composition. The variation is monotonous, but significantly non-linear. In the case of EHUT/BuPEH mixtures, the effect is much less visible: the EHUT solution (η/η 0 = 4.2) is only slightly more viscous than the BuPEH solution (η/η 0 = 4.0). Consequently, the viscosity of EHUT/BuPEH mixtures is nearly independent of composition.

Experimental

The synthesis of bis-ureas was described previously. 8 Solvents were used as received. Solutions were prepared under stirring or shaking at least 1 week prior to use. Heating at 50°C accelerated the dissolution process, but did not affect the measurements.

IR spectroscopy

Infrared spectra were recorded on a Nicolet Avatar 320 spectrometer in a KBr cell of 0.1 cm path length. The temperature was controlled with a heating device (P/N21525) from Specac. Consecutive measurements were separated by at least 10 minutes, to allow for thermal equilibration. Thermal expansion of the solutions was not corrected.

ITC

Heats of dissociation were measured using a MicroCal VP-ITC titration microcalorimeter. 12 The sample cell (1.435 cm 3 ) was filled with pure toluene. A relatively concentrated bis-urea solution in toluene was placed in a 0.295cm 3 continuously stirred (270rpm) syringe. A first 2µL aliquot was injected, without taking into account the observed heat, to remove the effect of solute diffusion across the syringe tip during the equilibration period. Subsequent aliquots of the solution (5µL) were automatically injected into the sample cell every 360s, until the syringe was empty.

Viscometry

Measurements were performed with Cannon-Manning semi-micro capillary viscometers. The solutions were not filtered.

Rheology

Measuring conditions for EHUT were described previously. 10 Measurements for DMHUT were performed on a strain controlled TA Instrument (ARES-LS1) equipped with a stainless steel Couette cell (diameters: 16.5mm -17mm; height: 13mm). In order to obtain reproducible results, the sample was first heated to 70°C for one hour and poured in the Couette geometry regulated at 20°C or 35°C. The measurements were performed after 20 minutes of equilibration. The protocol for each sample was the following: first, the sample was submitted to a strain sweep at 1 Hz in order to determine the viscoelastic regime, before reaching the non-linear regime the strain sweep was stopped in order to preserve the sample. The strain sweep was followed by a frequency sweep.

Conclusion

By using three different bis-ureas which self-assemble according to the same hydrogen bonding pattern, we have shown that it is possible to finely tune the properties of the resulting solutions. First, the critical temperature below which viscoelastic solutions are obtained (T**), varies linearly with the co-monomer composition and can thus be rationally adjusted. Second, the viscosity of the dilute solutions can also be finely tuned by changing the comonomer content. This clearly facilitates formulation, which is an important step as far as applications are concerned. Moreover, such a smooth behaviour is difficult to achieve with classical organogelators, which rely on crystalline fibre formation, because it is not a trivial task to find several compounds which can co-crystallize without morphological changes or self-sorting effects.

7

 7

Figure 1 .Figure 2 .

 12 Figure 1. Structure of studied bis-ureas

Figure 3 .

 3 Figure 3. Storage G' (•) and loss G" ( ) modulus versus frequency for bis-urea solutions in dodecane, at a concentration of 1g/L (2.3mM). (a) EHUT, 20°C; (b) DMHUT, 20°C; (c) DMHUT, 35°C.

Figure 4 .

 4 Figure 4. Ratio of absorbances at 3328 and 3300 cm -1 for 12.5mM toluene solutions of EHUT/DMHUT mixtures (100/0: ♦; 75/25: ; 50/50: •; 25/75: ; 0/100: ) versus temperature. Both heating and cooling runs are represented, showing the absence of hysteresis.

Figure 5 .

 5 Figure 5. Transition temperature between thick and thin filaments, for 12.5mM EHUT/DMHUT (•) and EHUT/BuPEH ( ) solutions in toluene, versus EHUT molar content.

Figure 6 .

 6 Figure 6. Relative viscosity (η/η 0 ), for 0.8mM EHUT/DMHUT (•) and EHUT/BuPEH ( ) solutions in toluene, versus EHUT molar content (25°C).

Figure S1 .Figure 2 .Figure 2 .Figure 2 .Figure 2 .Figure 3 .Figure 3 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure S1 .Figure S1 .

 S12222333456S1S1 Figure S1. Enthalpogram produced by injecting a 0.24mM toluene solution of DMHUT into toluene at 25 or 35 °C (5-µL aliquots). At 35°C, two transitions (C* and C**) are visible; at 25°C, the two transitions coincide.

Table 1

 1 Characteristics of bis-urea self-assembly in toluene Largest dimension of the fully extended monomer. b Diameter of the filament cross-section. c Derived from SANS measurement 8 of a 22mM solution in d 8 -toluene, at 22°C. d Mass per unit length of the filament. e Number of bis-urea molecules in the cross-section, assuming a repeat distance of 4.6Å. f Transition temperature between thick and thin filament structure, determined by FTIR measurement 8 of a 12.5mM solution in toluene. g Concentration corresponding to the transition between thick and thin filament structure at 25°C, determined by ITC (see Fig.2b).

	13,14

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Supramolecular Chemistry

Acknowledgements

C. Rossi and F. Dubut are thanked for their contribution to this project.

Notes and References

#

In the case of DMHUT, both transitions occur at very similar concentrations. This can be deduced from the value of the enthalpy change on Figure 2b, and is unambiguously proved by comparison to the result for the same experiment performed at 35°C (Figure S1). At this temperature, the 2 transitions are clearly separated. ‡ Insolubility of BuPEH in dodecane prevented its rheological characterization.


A Krajewska

M Smet

W Dehaen

H Radecka
email: hanna.radecka@pan.olsztyn.pl

Tetralactam-Modified Gold Electrodes for Amperometric Detection of Acrylic Acid

Keywords: acrylic acid, tetralactam, OSWV, gold electrodes acrylic acid, tetralactam, OSWV, gold electrodes

The presence of toxic acrylamide in a wide range of food products such as potato crisps, French fries or bread has been confirmed by Swedish scientists from Stockholm University. The neurotoxicity and possible carcinogenicity of this compound and its metabolites compels us to control them by quantitative and qualitative assays.

Exposing acrylamide to pH extremes results in its hydrolysis to acrylic acid or its salt. In this work we present the use of gold electrodes coated with SAMs containing tetralactam molecules and its precursor as active elements for voltammetric detection of acrylic acid in water solution. The host molecules have been immobilized on the electrode surface by covalent Au-S bond or by embedment method into thiol layer via hydrophobic and van der Waals interactions. Interactions with analytes were confirmed by Osteryoung square wave voltammetry (OSWV).

Introduction

Acrylamide is a well known neurotoxin and potential carcinogen [1,2]. High levels of this compound have been found in potato chips, French fries and several other common foods [3][4][5]. The first such report was announced by scientists from Stockholm University in 2002 [3]. Acrylamide forms in the reaction between reducing sugars such as glucose and amino acid asparagine. The Millard reaction mechanism has been proposed to account for its formation in high starch foods during cooking at high temperature [6][7][8].

Investigation has shown that high temperature and long heat treatment result in higher acrylamide content [6].

Among analytical methods used to determine acrylamide levels, expensive and time-consuming chromatographic techniques such as GC-MS [9], GC-MS-MS [10], HPLC-MS [11] and LC-MS-MS [5] predominate. Preparation of samples from food involves extraction using water or methanol and the clean-up step typically consists of a combination of several solid-phase extractions. GC-MS often needs the additionally laborious bromination step to form more volatile acrylamide derivative and increase selectivity of determination. Only a few examples of using different techniques for detection of this toxic compound exist.

Stobiecka and co-workers introduced a voltammetric sensor based on the reaction of hemoglobin with acrylamide [12]. This reaction leads to hemoglobin-acrylamide adducts formation that alters the electroactivity of hemoglobin. This biosensor showed good selectivity and sensitivity.

Exposing acrylamide to pH extremes results in its hydrolysis to acrylic acid or its salt. The most frequently used methods of acrylic acid determination also include GC [13] and HPLC [14]. Ignatov and coworkers prepared a biosensor in which respiratory activity of microbial cells was used for detection of acrylamide and acrylic acid [15]. Kleefisch and co-workers reported a sensor in which acrylamide and acrylic acid was detected at a gas-solid interface using an 'electronic nose'-type quartz crystal microbalance (QCM) sensor covered with a tetralactam active layer [16]. This system had not previously been tested at waterorganic interface.

Tetralactams belong to a wide group of neutral compounds able to complex anions by hydrogen bond formation [17,18]. These macrocycles have been used recently as the macrocyclic host for detection of carbonyl compounds. Hunter showed that such a macrocycle can complex p-benzoquinone and the recognition process can be achieved via a combination of π-π interaction and four hydrogen bond formations to the quinone oxygens. This phenomenon was confirmed by an 1 H NMR experiment performed in CDCl 3 [19]. A similar experiment performed in CD 2 Cl 2 and mixture of CD 2 Cl 2 /CD 3 OD at a 4:1 ratio showed interaction of these compounds with Br -, Cl -, weaker with NO 3 -and AcO - [20]. Tetralactam has been used in an electronic nose sensor to create a sensitive and selective active layer on the gold surface of quartz crystal electrodes and detect apple ester, 2-hexanal, s-limonesse and 2-heptanon [21,22]. Hydrogen bonds formation and π-π interaction were responsible for the formation of catenate and rotaxanes. Their synthesis was based on the action of supramolecular nucleophile that is formed from the molecular recognition of an anionic stopper by a tetralactam wheel [23][24][25].

Similar macrocycles containing pyridine fragments showed binding affinities for a diamide guest (Et 2 NC(O)(CH 2 ) 4 C(O)NEt 2 ). These interactions were investigated also in CDCl 3 by an 1 H NMR titration experiment [26].

In this paper we present the use of gold electrodes coated with SAMs containing tetralactam macrocycle and its acyclic derivative (Fig. 1) as active elements for voltammetric detection of acrylic acid in water solution. The host molecules have been immobilized on an electrode surface by covalent Au-S bond or by embedment method into the thiol layer via hydrophobic and van der Waals interactions. Influence of modification type on electrodes sensitivity has been investigated.

Experimental

Reagents and materials

1-Dodecanethiol (DDT), [Ru(NH 3 ) 6 ]Cl 3 , acrylic acid were purchased from Aldrich Chemie (Germany).

Potassium nitrate was purchased from POCh (Gliwice, Poland). Aqueous solution were prepared with deionized water (18.2 MΩcm resistivity) obtained with a Simplicity ® 185 Water System (Millipore, Molsheim, France).

Synthesis of receptors

Receptors 1, 2 and 3 have been synthesized in the Department of Chemistry, University of Leuven (Fig. 2).

Dodecyloxyisophthaloyl dichloride 1 [27], bisamine 2 [23], monoBoc derivative 6 [28] and benzyloxyisophthaloyl dichloride 9 [29] were obtained according to literature procedures. All other reagents were obtained commercially and used as received. NMR spectra were recorded on a Bruker Avance 300 or 400. To bisamide 7 (1.95 g; 2.0 mmol) CH 2 Cl 2 (5 mL) and trifluoroacetic acid (5 mL) were added and the resulting mixture was stirred for 1 h at room temperature. The mixture was poured into an aqueous saturated NaHCO 3 solution (100 mL) and the resulting suspension was extracted with CH 2 Cl 2 (3 x 70 mL). The combined organic layers were dried over MgSO 4 and the solvent was evaporated under reduced pressure, yielding bisamine 8 as a white solid in 83% yield. The spectral data were found to be identical to published data [23].

5-Dodecyloxyisophthaloyl bis(diamine)

Benzyl protected macrocyclic tetralactam 10 and sweeping the potential between -400 mV and -1200 mV (versus a Ag/AgCl reference electrode) with a scan rate of 100 mVs -1 till cyclic voltammograms no longer changed. The stop potential was -400 mV.

Modification of electrodes

Covalent modification

Directly after cleaning electrodes were soaked in 0.1 mM bis(tetralactam) disulphide solution (receptor 1) in ethanol/chloroform mixture (1/1 v/v) containing 1 mM dodecanethiol at room temperature for 30 min. Then after washing with ethanol and water the electrodes were stored in 0.01 M KNO 3 at room temperature until use.

Embedment modification

Directly after cleaning, the electrodes were soaked in 0.01 mM dodecanethiol solution in ethanol at room temperature for 30 min. After washing with ethanol the electrodes were soaked in 1 mM solution of receptor 2 or 3 in ethanol for 12 h. Then after washing with ethanol and water the electrodes were stored in 0.01 M KNO 3 at room temperature until use.

Before measuring, modified electrodes were stored in a solution containing 0.01 M KNO 3, pH = 5.0 for at least one day for conditioning.

Electrochemical measurement

All electrochemical measurements were performed with a potentiostat-galvanostat AutoLab (Eco Chemie, Utrecht, Netherlands) with a three-electrode configuration. Potentials were measured versus the Ag/AgCl reference electrode obtained from Bioanalytical Systems (BAS) West Lafayette, Indiana, USA.

Ag/AgCl wire was placed in a glass tube filled with 3 M NaCl. The tube was closed with a vycor plug to protect the inner solution from direct contact with the sample solution. A platinum wire was used as the auxiliary electrode. Cyclic voltammetry (CV) was performed and potential was cycled from +50 mV to -500 mV for 1.0 x 10 -4 M [Ru(NH 3 ) 6 ]Cl 3 with a scan rate of 500 mVs -1 . Osteryoung square-wave voltammetry

Results and Discussion

Synthesis of receptors

An open chain bisamine 3 (receptor 3) was synthesized by reaction of dodecyloxyisophthaloyl dichloride 1 [27] with a large excess of bisamine 2 [23] (Fig. 2). Bisamine 3 was then cyclized to receptor 2 under high dilution conditions by reaction with isophthaloyl dichloride (4). For the synthesis of receptor 1, we first started the preparation of bisamine 8 on the basis of the literature [23]. However, it was found that largescale preparation of the latter compound proceeded more conveniently via the protected precursor 7, which was readily obtained by the reaction of equivalent amounts of isophthaloyl dichloride (4) and the monoprotected derivative 6 [28]. After deprotection, the bisamine 8 was allowed to react with benzyloxyisophthaloyl dichloride 9 [29] under high dilution conditions, yielding macrocyclic lactam 10.

Hydrogenolytic removal of the benzyl group followed by Williamson etherification afforded the alkenylsubstituted analog 12 via intermediate phenol 11 -which was not isolated. After the radical addition of thioacetic acid, thioester 13 was isolated which, upon smooth basis hydrolysis in the presence of oxygen, gave rise to disulfide 14 or receptor 1.

Formation and characteristics of tetralactam SAMs

Our paper presents two methods of immobilization of SAMs on gold electrode surfaces. The covalent method of modification involved one step in which 1-dodecanethiol and bis(tetralactam) molecules (receptor 1, Fig. 1) mixed in one solution were immobilized simultaneously on gold. Such a prepared mixed monolayer consisted of ionophore molecules covalently bonded to the electrode surface and dedecanethiol molecules.

This procedure was used to prepare a dense monomolecular layer and avoid a pinholes formation. Dodecanethiol was chosen because of its ability to create a well-ordered monolayer and serve as an inert background for receptor molecules. Chain lengths of receptor tetralactam and dodecanethiol were similar, which helps create the ordered monolayer.

The embedment method involved two steps. Firstly, electrodes were modified only by 1-dodecanethiol to form a homogenous liphophilic monolayer on the surface. Then the macrocycle possessed one liphophilic side chain (receptor 2 or 3, Fig. 1) and was immobilized via hydrophobic and van der Waals interactions [30,31]. The layer formed by this procedure was rather bimolecular.

The monolayer prepared by the covalent method was better stabilized than that prepared by embedment. The first method assumed the creation of a covalent bond, but hydrophobic interactions between liphophilic side chain of the ionophore and liphophilic chains of 1-dodecanethiol also play some role in SAM stabilization. We found that electrodes modified by this method are stable during CV measurements performed in the wide potential window (-500 to +50 mV). Mirsky and co-workers [32] postulated desorption of alkanethiol monolayer at electrode potentials -300 and -600 mV in the absence of alkanethiol in the bulk solution at pH=5.7. The loss of monolayer stability was observed when negative potential was applied for longer than 15 min. In our work negative potential was also applied to the modified electrodes but time of exposure was considerable lower: less than one minute in the case of CV (scan rate CV 500 mV/s) and OSWV (step potential 5 mV, frequency 100 Hz) and about 3 min. in the case of impedance measurement.

Because of this we did not observe any influence of negative potential applying and monolayer stability was kept.

In the case of embedment modification receptor molecules are bound to the electrode surface by weaker forces than in the case of covalent immobilization, but references [30,31] give examples of using the same procedure to obtain stable and reproducible SAMs.

Both modification procedures were monitored with OSWV. In order to display the role of tetralactam host in SAMs the gold electrodes modified only with 1-dodecanethiol was used. These electrodes were measured with cyclic voltammetry (CV) and Osteryoung square wave voltammetry (OSWV) with [Ru(NH 3 ) 6 ] 3+ as redox marker. Obtained voltammograms were compared with results obtained for electrodes coated additionally with tetralactam by both methods of modification (Fig. 1 supplementary material). In the event of embedment technique of modification for electrodes coated with a mixed layer the peak current of reduction/oxidation process of [Ru(NH 3 ) 6 ] 3+ was much lower in comparison with thiol monolayer (Fig. 1A supplementary material). Access of [Ru(NH 3 ) 6 ] 3+ to the surface of electrodes modified by embedment was more hindered probably because of the bilayer structure of this SAM. Covalent modification led to monolayer creation. In this case for electrodes coated with a mixed layer was observed higher peak current in comparison with electrodes modified with thiol monolayer (Fig. 1B supplementary material). The created mixed layer was less ordered than that formed only by one type of thiol molecules and access of redox marker ions was facilitated.

Comparison of both investigated mixed SAMs showed that for electrodes coated with embedment lower peak current was observed. Access of redox marker molecules to the bilayer-modified electrode surface were more hindered than to the surface of electrodes modified covalently with mixed monolayer.

Similar results were obtained for electrodes modified with macrocyclic polyamine -also by covalent and embedment methods [30,31], where the same correlation appeared.

For investigated SAMs EIS experiment was also performed. The results were displayed as the relation of the The constant phase element (CPE, Q dl ) was used instead of the ideal capacitor in order to improve the fitting and minimize errors (typical plot of fitting is shown in Fig. 7 in the supplementary material). The CPE expressed the double-layer capacitance and its use is caused by a microscopic roughness or heterogenity of gold electrode surface [33].

The impedance of CPE is given by:

Z CPE =1/(Y 0 jω) n
where Y 0 is CPE constant, j = (-1) 1/2 , ω = 2πf being f the frequency of the applied ac potential and n is a fractional parameter equal from 0.5 (for an ideally porous electrode) to 1.0 (for a perfectly smooth electrode).

When n=1, Y 0 ≡C dl (capacity of double layer) and purely capacitive behavior is obtained. A small deviation from 1.0 (n > 0.9) was observed in this study, which suggests that Y 0 parameter can be treated as a capacity of double layer. DDT monolayer prepared from 0.01 mM solution was higher to those obtained for embedment modifications.

It showed that embedment modification leads to the formation of ticker SAM in comparison with DDT layer.

These results are in good agreement with results obtained for OSWV (Fig. 1 supplementary material).

Sensitivity of tetralactam SAMs toward acrylic acid

The determination of acrylic acid by gold electrodes modified with tetralactam by covalent and embedment methods has been examined using two techniques: CV and OSWV in 0,01 M KNO 3 . Redox marker [Ru(NH 3 ) 6 ]Cl 3 was most suitable for the investigated system. The negatively charged [Fe(CN) 6 ] 3+/4+ was shown to be irreversible in cases of investigated modifications. Moreover, this marker demands higher concentration of supporting electrolyte (0.1 M), which can decrease interaction between the host and anionic guest. Rutenium complex can be used in lower concentrations of supporting electrolyte (0.01 M). Due to the reasons noted above, [Ru(NH 3 ) 6 ] 3+ was chosen.

The interaction between a tetralactam host immobilized on Au electrodes surface by covalent and embedment methods and anion of acrylic acid were investigated by voltammetric techniques CV and OSWV Electrodes modified by covalent method had similar detection limit, which was 1.0 x 10 covalent modification it was from 1.0 x 10 -5 to 2.5 x 10 -4 , with slope -42.8 %/decade (obtained from the plot I p /I p,0 x 100% vs log c) and regression coefficient 0.976. In the case of embedment it was from 1.0 x 10 -5 to 1.0 x 10 -3 M with slope -10.6 %/decade and regression coefficient 0.930.

A stronger response toward acrylic acid was observe with electrodes modified by covalent method (Fig. 3, Fig. 2 supplementary material) in relation to those modified with embedment (Fig. 4). The highest acrylic acid concentration studied (1.0 x 10 -3 M) caused 67.5% current decrease in OSWV in the case of covalent modification and 21.3% for electrodes modified by embedment. Electrodes modified with acyclic derivative (receptor 3) were also investigated. These electrodes prepared by embedment and measured in the same conditions of pH showed inconsiderable response toward acrylic acid (Fig. 6 supplementary material).

The cyclic voltammetry showed lower sensitivity as compared with square wave voltammetry. Important advantage of OSWV is the ability to suppress the background and because of that is more sensitive and more suitable for quantitative measurements. The results obtained by CV and OSWV are presented in the Fig. 3 and Fig. 2 supplementary material.

Electrochemical impedance spectroscopy (EIS) was used to detect acrylic acid with electrodes modified by covalent method. Results obtained with EIS were with good agreement with results obtained with OSWV. The observed range of response was similar in both cases. Results obtained with EIS are presented in Parameters such as solution resistance and the constant phase element should remain almost constant for measurements performed in the absence and in the presence of a faradaic process. Determination of these parameters in the absence of a reaction and using them as initial estimates in the presence of the reaction helps to reduce the error in the fit. However, faradaic impedance spectroscopy technique using a redox probe molecule and measured at the formal potential of the redox probe can reveal a dependence of electron transfer resistance in the SAM during complex formation. Formal potential of the redox marker was chosen as a potential at which the data were collected on the basis of references [38][39][40].

As a control experiment electrodes modified only with 1-dodecanethiol were used (Fig. 4 supplementary material). These electrodes showed only negligible response toward the analyte in the solution pH=5.0. This proved that only the tetralactam host presence was responsible for recognition process.

Electrodes modified with acyclic derivative (receptor 3) also were investigated. These electrodes, prepared by embedment technique and measured in the same conditions of pH, showed inconsiderable response toward Deleted: The complex impedance was displayed as the sum of the real (Zre) and imaginary (Zim ) components and presented as Nyquist plots (Zim vs Zre). acrylic acid (Fig. 6 supplementary material). This suggested that only interaction between macrocyclic tetralactam and acrylic acid is sufficient.

Mechanism of response

In the present paper the interaction between acrylic acid and tetralactam molecules immobilized on the surface of gold electrodes either by the covalent or embedment methods was investigated. CV, OSWV and electrochemical impedance spectroscopy measurements were performed in the presence of 0.01 M KNO 3 used as supporting electrolyte. Appropriate pH values (3.0, 5.0 and 6.2) were obtained by the addition of KOH or HNO 3 solutions. Because of interference and suppressing of response no buffer solution was used.

At pH=5.0 analyte existed in the solution in 85% as anionic form and in 15% as neutral form, whereas tetralactam existed in the layer on the electrode surface as a neutral compound. This ionophore and analyte in ionic or neutral form can interact by hydrogen bonds formation. Tetralactam macrocycle have four amide fragments, that can form strong H-bonds and can serve both as donors and acceptors of such bonds. π-π interactions between double bonds of analyte and aromatic rings of host molecule also can play some role [16].

Geometry optimization of a number of different conformations of the macrocycle in vacuum using CaChe

Workspace programme (CaChe Workststem Pro Version 7.5.0.85) and AM1 geometry procedure (MOPAC 2002 Version 2.5.3, J. J. P. Stewart, Fujitsu Limited, Tokyo, Japan) showed that most favorable conformation of the host molecule is when all four amide -NH group (acceptors of H-bond) are oriented inside the macrocycle cavity (-185.10 kJ). Conformation with one -NH group oriented outside the cavity is slightly less favorable (-172.77 kJ). These results are in good agreement with similar calculations in CHCl 3 conducted by Kleefisch and coworkers [16] and structures obtained by X-ray crystallography [41]. These results indicate the strong H-bond accepting character of the investigated macrocycle cavity.

Acrylic acid in dissociated form possess stronger electron donor characters and, because of that, stronger affinity to the tetralactam cavity in comparison with neutral form. That suggests that observed response was caused more by anionic than by neutral form of analyte.

In order to support the above conclusion OSWV and EIS experiments in solution pH=6.2 was conducted. In these conditions about 100% of analyte existed in anionic form and the expected response of the sensor should be improved. However, electrodes measured in solution at higher pH showed considerably lower response for acrylate anion (Fig. 5, Fig. 5 supplementary material). A possible explanation for this phenomenon can be the interaction of the ionophore with OH -ions, which can also be strong H-bond donors and have high affinity to the tetralactam cavity.

Measurement conducted by EIS in pH=3.0 (when analyte exist in solution in neutral form) showed no response of electrodes modified with covalent method towards acrylic acid. These results showed that pH=5.0 was optimal for detection of the analyte and confirmed the conclusion that observed interaction occurs between the ionophore and anionic forms of analyte.

The working principal of the sensor presented suggests that it can be assigned to "ion-channel" or "ion-channel-mimetic" sensors. Umezawa and co-workers who have introduced these types of sensors have distinguished two types of such sensors according to mechanism of response: intermolecular and intramolecular ion-channel sensors [42][43][44]. In the first group electrostatic ion-ion interaction (electrostatic attraction or repulsion) and physical blocking of the intermolecular voids prevents access of redox marker to the electrode surface. Binding ionic analytes to the synthetic receptor layer changes the total charge of the layer and its permeability for marker ions. In intramolecular ion-channel sensors, access of markers to electrode surface through the intramolecular cavity of receptors can be blocked by the formation of inclusiontype complexes with an analyte.

In measuring conditions the acrylate anion can interact with neutral tetralactam molecules to form a negatively charged complex. This may give negative charge to the layer and increase attraction between the electrode and positively charged redox marker [Ru(NH 3 ) 6 ] 3+ . However, a sensor reported decreasing of peak current. Probably the physical blocking of intermolecular spaces prevented access of marker ions to the electrode and played a decisive role in the generation of the investigated sensor response. This can be explained by the idea of an intermolecular ion-channel mechanism [42][43][44].

Supramolecular complex formations not only block reaction of electron transfer between the electrode and marker molecules but also affect its kinetics. Reversibility of the reduction/oxidation process of [Ru(NH 3 ) 6 ] 3+ decreased. So the maximum of peak current observed by OSWVs decreased and shifted to more negative potential values. In the case of covalent modification investigated in this work the highest acrylic acid concentration studied (1.0 x 10 -3 M) caused 67.5% current decrease in OSWV and a slight shift of peak potential. In the case of electrodes modified by embedment method lower current decrease (21.3%), but higher negative shift of peak potential was observed. This phenomenon can be connected with different characters of two investigated monolayers. The embedment technique provide more flexibility of ionophore molecules in comparison with the covalent method, which can entail different characteristics of a complex Gold electrodes coated with host molecule by covalent method showed higher sensitivity than those coated by embedment modification (Figs. 3,4). Embedment technique provided higher flexibility of prepared layer. Macrocycle interacts with thiol layer via non-covalent interaction and has the possibility to take the most suitable conformation for interaction with analyte. The covalent technique did not provide such "conformational freedom". Because of the above reasons, higher sensitivity for embedment modification was expected. However, obtained results were different.

Measured tetralactam is 32-membered macrocycle which possess six benzene ring fragments and, as the CPK model showed, is rather rigid and does not have the ability to adopt its conformation. Another possible explanation can be the difference in structure of two investigated layers. The covalent technique makes it possible to create a monolayer containing tetralactam molecules. In the case of embedment technique a bilayer is formed and ionophore molecules recede from the electrode surface, which can explain the decrease of sensitivity.

In this case a crucial factor can also be the density of electrode surface coverage by the host molecules, which for the covalent method can be higher. Investigated tetralactam after modification is covalently bonded to the electrode surface via one side chain. Embedment assumes the attachment of the macrocycle by hydrophobic and van der Waals interactions between the alkanethiol monolayer and aliphatic side chain of host molecule.

Measured tetralactam possess only one side chain and because of that such prepared SAMs were less stable and less reproducible in comparison with the covalent method.

A similar comparison of these two techniques of immobilization was made using polyamine macrocycle [30,31]. This 30-membered macrocycle possess two aromatic rings as fragments of macrocycle. Electrodes modified with this bigger and significantly more flexible molecule by embedment showed better sensitivity than those coated by covalent technique. Moreover, these macrocycles formed stable and reproducible SAMs via six aliphatic side chains versus the investigated tetralactam that possessed one side chain. The interaction between the tetralactam host and acrylate guest could be observed using Osteryoung square wave voltammetry (OSWV) and electrochemical impedance spectroscopy (EIS).

The covalent method of host immobilization on the gold electrode surface was more suitable than embedment.

Electrodes modified by covalent technique were characterized by better sensor-to-sensor repeatability in comparison to those modified by embedment.

Faradaic impedance spectroscopy as well as CV and OSWV was used to investigate reported systems and based on results obtained with these methods intermolecular ion channel mechanism of response was proposed.

F

  was performed in the same potential range with a step potential of 5 mV, a square-wave frequency of 100 Hz, and amplitude of 25 mV. The dependence of the sensor responses on the concentration of analytes was expressed as the currents at the peak potential in OSWVs for [Ru(NH 3 ) 6 ] 3+ measured in a solution containing 0.01 M KNO 3 . Electrochemical impedance spectroscopy (EIS) was performed within the frequency range of 0.1 Hz to 10 kHz at the formal potential of the redox couple [Ru(NH 3 ) 6 ] 2+/3+ (-0.2 V) with ac amplitude of 10 mV.

Deleted: filling the voids between

  imaginary (-Z'') versus the real (Z') impedance components plotted for various frequencies (the Nyquist plots -Z'' vs Z'). The equivalent circuit model used to fit the experimental data is shown in Fig.5E. It consists of resistance solution Rs, constant phase element Q dl , charge transfer resistance R ct and Wartburg element Z w .

[ 34 -

 34 37]. The measurements were performed in a pH=5.0 solution. Electrodes modified with covalent technique showed different peak current (from -0.39 to -1.07 µA) and peak potential (from -184.0 to -343.0 mV) in 0.01 M KNO 3 solution containing 0.1 mM [Ru(NH 3 ) 6 ]Cl 3 . Electrodes modified with embedment method also showed differentiation in peak maximum parameters obtained in 0.01 M KNO 3 solution containing 0.1 mM [Ru(NH 3 ) 6 ]Cl 3 . For modification with receptor 2, peak current values were in range from -0.51 to -0.67 µA and peak potential from -262.0 to -292.0 mV. For modification with receptor 3 peak current was in range from -1.03 to -1.68 µA and peak potential -143.0 to -163.0 mV. Because of this parameter of relative decreasing of peak current was used to evaluate their response. The ratio of OSWV peak current in the presence of different concentrations of acrylic acid (I p ) to that in the absence of acrylic acid (I p,0 ) (I p /I p,0 x 100%) was plotted versus the acrylic acid concentration. Even though the modified electrodes studied showed different maximum current in 0.01 M KNO 3 solutions, all of them modified with the same method displayed similar sensitivity toward acrylic acid estimated with using the relative current decreasing (I p /I p,0 x 100%).

Fig. 5 .

 5 Fig. 5. The equivalent circuit model used to fit the experimental data is shown in Fig. 5E. It consists of resistance solution R S , constant phase element Q dl , charge transfer resistance R ct and Wartburg element Z w .

  increasing acrylate anion concentration the Faradaic current of redox reaction decreased for electrodes modified either by the covalent or embedment methods. The size of tetralactam cavity estimated by AM1 MOZYME geometry optimization (5.845 Å -the smallest width) but also estimated from Corey-Pauling-Koltun atomic (CPK) model (6.4 Å) is large enough to hold the analyte (5.1 Å -CPK model) but not large enough for the marker [Ru(NH 3 ) 6 ] 3+ (6.4 Å[45]) to penetrate through. This also confirms the conclusion of investigated sensor work according to the intermolecular ion-channel sensor mechanism.

Conclusions

  It was proved that tetralactam macrocycle can be used as the host molecule of chemically modified gold electrodes destined for detection of acrylic acid in water solution.

Figure 1 .

 1 Figure 1. Structural formulas of ionophores used for modification of electrodes with covalent (A) and embedment methods (B, C).

Figure 2 .

 2 Figure 2. Synthetic route to obtain receptors 2 and 3 (A) and receptor 1 (B).

Figure 3 .

 3 Figure 3. (A) The OSWV curves for [Ru(NH 3 ) 6 ] 3+ measured with electrodes modified by covalent method (receptor 1) in the presence of various concentrations of acrylic acid. The electrolyte composition: 0.01 M KNO 3 , pH = 5.0, 0.1 mM [Ru(NH 3 ) 6 ] 3+ . Step potential: 5 mV, square-wave frequency 100 Hz, and squarewave amplitude 25 mV. The concentration of the analyte was: (A) 0, (B) 1.0 × 10 -6 , (C) 1.0 ×10 -5 , (D) 2.5 × 10 -5 , (E) 5.0 × 10 -5 , (F) 1.0 × 10 -4 , (G) 2.5 x 10 -4 , (H) 5.0 x 10 -4 , (I) 1.0 x 10 -3 M. (B) The ratio of OSWV peak current in the presence of different concentrations of acrylic acid (I p ) to that in the absence of acrylic acid (I p,0 ) as a function of the acrylic acid concentration. The currents were measured at the peak potential in OSWV curves in the solution with no analyte E p,0 = -243 mV, n=3, 5.4%<SD<15.0%.

Figure 4 .

 4 Figure 4. The ratio of OSWV peak current with electrodes modified by embedment technique with receptor 2 in the presence of different concentrations of acrylic acid (I p ) to that in the absence of acrylic acid (I p,0 ) as a function of the acrylic acid concentration. The currents were measured at the peak potential in OSWV curves in the solution with no analyte E p,0 = -292 mV, n=3, 3.6%<SD<7.7%.

Figure 5 .

 5 Figure 5. Impedance plots of 0.1 mM [Ru(NH 3 ) 6 ] 3+ measured in the absence and in the presence of different concentrations of acrylic acid in the solution of 0.01 M KNO 3 (A) pH=3.0, (B) pH=5.0, (C) pH=6.2; (D) Plot of relative change of R ct with concentration of analyte for solutions pH=3.0 (▲), 5.0 (■), 6.2 (•), (E) Equivalent circuit used to fit the experimental data: R S represents the solution, Q dl -constant phase element, R ct -charge transfer resistance, Z W -Wartburg element.

  Model of complex formation between tetralactam and acrylate (CaChe Workststem Pro Version 7.5.0.85, Fujitsu Limited, Tokyo, Japan).

Figure 1 .Figure 1 .

 11 Figure 1. Structural formulas of ionophores used for modification of electrodes with covalent (A) and embedment methods (B, C).

Figure 1 .

 1 Figure 1. Structural formulas of ionophores used for modification of electrodes with covalent (A) and embedment methods (B, C).

4 AFigure 2 .

 42 Figure 2. Synthetic route to obtain receptors 2 and 3 (A) and receptor 1 (B).

Figure 2 .Figure 3 .

 23 Figure 2. Synthetic route to obtain receptors 2 and 3 (A) and receptor 1 (B).

Figure 3 .Figure 4 .Figure 5 .Figure 5 .Figure 5 .Figure 5 .Figure 5 .Figure 1 . 3 Figure 2 . 4 Figure 3 . 5 Figure 4 . 3 Page 38 of 42 URL 6 Figure 5 . 6 Page 39 of 42 URL 7 Figure 6 .Page 40 of 42 URL 8 Figure 7 . 9 Table 1 .

 345555513243543426564276428791 Figure 3. (A) The OSWV curves for [Ru(NH 3 ) 6 ] 3+ measured with electrodes modified by covalent method (receptor 1) in the presence of various concentrations of acrylic acid. The electrolyte composition: 0.01 M KNO 3 , pH = 5.0, 0.1 mM [Ru(NH 3 ) 6 ] 3+ . Step potential: 5 mV, square-wave frequency 100 Hz, and square-wave amplitude 25 mV. The concentration of the analyte was: (A) 0, (B) 1.0 × 10 -6 , (C) 1.0 ×10 -5 , (D) 2.5 × 10 -5 , (E) 5.0 × 10 -5 , (F) 1.0 × 10 -4 , (G) 2.5 x 10 -4 , (H) 5.0 x 10 -4 , (I) 1.0 x 10 -3 M.(B) The ratio of OSWV peak current in the presence of different concentrations of acrylic acid (I p ) to that in the absence of acrylic acid (I p,0 ) as a function of the acrylic acid concentration. The currents were measured at the peak potential in OSWV curves in the solution with no analyte E p,0 = -243 mV, n=3, 5.4%<SD<15.0%.

  Diamine 2 (21.0 g; 65.2 mmol) was dissolved in CH 2 Cl 2 (100 ml) and triethylamine (3.2 mL) was added. 5-Dodecyloxyisophthaloyl dichloride (4.50 g; 11.6 mmol) 1 was similarly dissolved in CH 2 Cl 2 (100 mL) and added dropwise to the diamine solution during 2 h with stirring under argon. The solvent was evaporated under reduced pressure and the dissolved residue was purified by column chromatography on silica with CH 2 Cl 2 /ethyl acetate 7:1 as the eluent yielding 3 (2.9 g; 26.3%).1 H NMR (δ, CDCl 3 , 400 MHz) 7.97 (s, 1H), Diamine 3 (0.91 g; 0.95 mmol) and Et 3 N (0.29 g; 2.8 mmol) were dissolved in CH 2 Cl 2 (20 mL). Isophthaloyl dichloride 4 (0.19 g; 0.95 mmol) was dissolved in a separate volume of CH 2 Cl 2 (20 mL). Both solutions were slowly added with an infusion pump during 4 h to a stirred volume of CH 2 Cl 2 (1.2 L) at room temperature. After complete addition, stirring was continued for 10 h at room temperature, after which the solvent was evaporated under reduced pressure. The residue was subjected to column chromatography on SiO 2 with CH

	Bisamine 8
	7.57 (s, 2H), 7.43 (s, 2H), 7.00 (s, 4H), 6.85 (s, 4H), 4.07 (t, 2H), 3.45 (s br, 4H), 2.19 (m, br, 16H), 2.14 (s,
	12H), 1.81 (m, 2H), 1.54-1.47 (m, 16H), 1.25-1.27 (m, 20H), 0.87 (t, 3H). ESI MS (m/z) 960 MH + .
	Macrocyclic tetralactam 5 (receptor 2)
	F o F o
	r r
	P P
	e e r e e r
	R e v i e w R e v i e w
	3 (receptor 3)	O n O n l y l y

2 Cl 2 /ethyl acetate 4:1 as the eluent yielding 5 as a white solid in 16 % yield.

1

H NMR (δ, CDCl 3 /CH 3 OD 19:1, 300 MHz) 8.31 (m, br, 1H), 8.16-8.13 (m, br, 2H), 7.85 (s, br, 1H), 7.66-7.65 (m, br, 3H), 6.99 (s, 8H), 4.09 (t, 2H), 2.45 (m,br, 4H), 2.34 (m, br, 4H), 2.27 (m, br, 16H), 2.17 (s, 12H), 1.82 (m, 2H), 1.65 (m, br, 4H), 1.52(m, br, 4H), 1.27-2.25 (m, br, 20H), 0.88 (t, 3H). ESI MS (m/z) 1089 MH + .

Boc-protected bisamide 7

Mono-boc-protected derivative 6 (2.1 g; 5.0 mmol) and Et 3 N (0.50 g; 5.0 mmol) were dissolved in CH 2 Cl 2 (60 mL) and added dropwise over 30 min to a stirred solution of isophthaloyl dichloride 4 (0.50 g; 2.5 mmol) in CH 2 Cl 2 (100 mL) at room temperature. After complete addition, the solution was stirred for another 2 h and the solvent was evaporated under reduced pressure. The target bisamide 7 was obtained after column chromatography on SiO 2 with CH 2 Cl 2 /ethyl acetate 4:1 as the eluent as a white solid in 76% yield.

1

H NMR (δ, CDCl 3 , 300 MHz) 8.52 (s, 1H), 8.22 (m, br, 2H), 7.74 (m, br, 2H), 6.69 (s, 4H), 6.96 (s, 4H), 6.81 (s, br, 1H), 5.97 (s, br, 2H), 2.19 (m,br, 16H), 2.06 (s, 12H), 1.55-1.39 (m, br, 16H), 1.39 (s, 18H). ESI MS (m/z) 975 MH + .

Preparation of working electrodes

  (0.49 g; 1.6 mmol) was dissolved in another volume of CH 2 Cl 2 (15 mL). Both solutions were added dropwise at room temperature to a stirred volume of CH 2 Cl 2 (1.5 L) over 4 h. Stirring was continued for 10 h at room temperature. The solvent was evaporated under reduced pressure and the macrocyclic tetralactam 10 was obtained as a white solid after column chromatography on SiO 2 with CH 2 Cl 2 /ethyl acetate

	wet 0.3 and 0.05 µm alumina slurry (Alpha and Gamma Micropolish; Buehler, Lake Bluff, Illinois, USA) on a
	Thioacetate 13 microcloth pad (BAS) and in a sonicator (30 sec.), then electrochemically by dipping in 0.5 M KOH solution
	Undecenyloxy derivative 12 (80 mg; 0.075 mmol) was dissolved in a 1:1 mixture of CH 2 Cl 2 and methanol (50
	mL). Thioacetic acid (57 mg; 0.75 mmol) and AIBN (2 mg) were added and the resulting solution was
	irradiated at room temperature with a UV lamp (375 nm, 50 Watt) under argon atmosphere for 12 h. The
	solvent was evaporated under reduced pressure and the residue was dissolved in a 9:1 mixture of
	CH 2 Cl 2 /methanol (approximately 0.5 mL). This solution was added drop wise to a stirred volume of
	diisopropyl ether (20 mL). The precipitate was filtered and washed with diisopropyl ether (2 x 5 mL) and
	Bisamine 8 (1.2 g; 1.6 mmol) and Et 3 N (0.48 g; 4.8 mmol) were dissolved in CH 2 Cl 2 (15 mL) and dicarbonyl dried in vacuum, yielding the title compound 13 in 86% yield. 1 H NMR (δ, CDCl 3 , 300 MHz) 8.21 (tr, br,
	F o r P 1H), 8.14 (d, br, 2H), 8.13 (d, b, 2H), 7.72, s, br, 1H), 7.66 (m, b, 3H), 7.31 (s, 2H), 6.97(s, 8H), 4.10 (t, br, 2H), 2.85 (t, 2H), 2.30-2.28 (m, br, + s, 11H), 2.18-2.16 (s, 16H), 1.86-1.82 (m, br, 4H), 1.67-1.64 (m, br, 8H), 1.56-1.53 (m, br, 14H), 1.30-1.26 (m, br, 12H). ESI MS (m/z) 1150 MH + . Receptor 1 (14) Thioacetate 13 (50 mg; 0.044 mmol) was dissolved in a 2:1 mixture of THF and methanol (5 mL). The F o r P dichloride 9 Undecenyloxy substituted macrocyclic tetralactam 12 Benzyl protected tetralactam 10 (100 mg; 0.099 mmol) was dissolved in a 1:1 mixture of CHCl 3 and ethanol e e r resulting solution was placed in an air tightvial closed with a septum. Argon was bubbled through the solution for 15 minutes via a syringe to remove oxygen. An aqueous solution of NaOH (1 mL, 1 M) was added via a syringe while keeping the reaction mixture under argon. The mixture was left for 10 h at room temperature, after which diluted HCl was added (1.1 mL, 1 M) via a syringe. The solvents were evaporated under reduced e e r
	(12 mL). Palladium on carbon (10%) (0.5 g) was added and the resulting suspension was shaken vigorously for 15 h under hydrogen atmosphere. The suspension was filtered over celite and the solids were washed with ethanol (2 x 5 mL). The combined filtrates were evaporated under reduced pressure and the residue containing phenol 11 was dissolved in DMF (10 mL). 11-Undecenyl bromide (46 mg; 0.20 mmol) and K 2 CO 3 (28 mg; 0.20 mmol) were added and the resulting suspension was heated under continuous stirring at 80°C under argon atmosphere. After cooling to room temperature, the solvent was evaporated under reduced pressure and CH 2 Cl 2 (10 mL) and water (10 mL) were added to the residue. After vigorous shaking, the organic layer was separated, dried over MgSO 4 and evaporated under reduced pressure. The title compound 12 was obtained as R e v i e w pressure and the residue was treated with a 19:1 mixture of CH 2 Cl 2 and methanol (approximately 2 mL) to dissolve the organic material. The resulting solution was filtered and the filtrate was added drop wise to a stirred volume of diethyl ether (10 mL) to precipitate 14 which was collected by filtration, washed with diethyl ether (2 x 2 mL) and obtained as a white solid in 45% yield. 1 H NMR (δ, CDCl 3 /CH 3 OH 19:1, 300 MHz) 8.30 (s, br, 2H), 8.16 (s, 8H), 8.09 (s, br 4H), 7.82 (t, br, 2H), 7.65 (tr + d,br, 6H), 6.98 (s, 16H), 4.08 (t, br, 4H), 2.68 (t, 4H), 2.34-2.32 (m, br, 16H), 2.17-2.15 (m br + s, 40H), 1.86-1.82 (m, br, 8H), 1.66-1.63 (m, br, 16H), 1.49-1.28 (m, br, 44H). ESI MS (m/z) 1129 [(MNa) 2 ] 2+ . R e v i e w
	a white solid after column chromatography over SiO 2 with CH 2 Cl 2 /ethyl acetate 4:1 as the eluent in 72% yield. 1 H NMR (δ, CDCl 3 /CH 3 OD 19:1, 300 MHz) 8.36 (s, 1H), 8.14 (d, 2H), 7.86 (s, 1H), 7.66 (s, 2H), 7.64 (t, 1H), 6.99 (s, 8H), 5.82-5.71 (m, 1H), 5.01-4.90 (m, 2H), 4.09 (t, 2H), 2.33-2.03 (m, 32H), 1.81 (m, 4H), 1.65-1.52 (m, 12H), 1.30-1.32 (m, 12H). ESI MS (m/z) 1072 MH + . O n l y Gold disk electrodes (2 mm 2 area, Bioanalytical Systems (BAS) West Lafayette, Indiana, USA) were O n used for all experiments. Before modifications the electrodes were cleaned mechanically by polishing with l y	Deleted: physically

4:1 as the eluent 21% yield.

1

H NMR (δ, CDCl 3 , 300 MHz) 8.37 (s, 1H), 8.16 (d, 2H), 7.94 (s, 1H), 7.83-7.77 (m, 4H), 7.71 (s, 2H), 7.45-7.32 (m, 5H), 6.98 (s, 8H), 5.18 (s, 2H), 2.78 (s, 6H), 2.53 (s, 6H), 2.30-2.33 (m, 6H), 2.16 (s, 8H), 1.62-1.51 (m, 12H). ESI MS (m/z) 1033.6 MNa + .

Table 1

 1 supplementary material showed values of Y 0 constant for investigated SAMs. The value of this parameter decreases with increases in the thickness of the SAM. The value of Y 0 for DDT monolayer prepared from 1.0 mM solution was similar to that obtained for covalent modification. It can be explained by the fact that receptor 1 used for this modification had a side chain with similar length (C11) to the length of dodecanethiol chain (C12) and these two monolayers could have comparable thickness. The value of Y 0 for

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y

  -5 M, but slightly different linearity region in comparison with embedment modification with receptor 2. In the case of

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y

  Lasia, A. in Conway, B. E.; Bockris, J.; White, R.E. (Edts.), Electrochemical Impedance Spectroscopy and Its Applications, Modern Aspects of Electrochemistry. Kluwer Academic/PlenumPublishers, New York,

	o F o [33] F o
	r r r
	P P P
	e e r e e r e e r
	R e v i e w R e v i e w R e v i e w
	O n O n O n
	l y l y l y

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Supramolecular Chemistry

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu

Acknowledgements

This work was supported by European grant COST D31/0021/05 and grants from the Polish Ministry of Science and Higher Education No. 19/COS/2006/3, No. 2PO6T08827 and Institute of Animal Reproduction and Food Research of Polish Academy of Sciences.


Estelle Métay

Marie-Christine Christine Duclos

Stéphane Pellet-Rostaing

Marc Lemaire

Jürgen Schulz

Ramu Kannappan

Christophe Bucher

Eric Saint-Aman

Carole Chaix

Conformational analysis and anion binding properties of ferrocenyl-calixarene receptors

Keywords: calixarene, ferrocene, intramolecular H-bonding, anion recognition, cyclic voltammetry

come

∆E = (E pa + E pc )/2; ∆E 1/2 = (E 1/2 ) L -(E 1/2 ) [L . (H 2 PO 4 -) 2 ]
associating calixarene as modular platform, urea units as anion binding moieties and ferrocene redox active reporters. Urea fragments have already been introduced into ppositions of calixarene skeleton mostly through direct linkage to elaborate supramolecular capsular materials [4][5][6][7][8][9] and anions or ditopic receptors. [10][11][12][13][14][15][16][17][18][19][20][21][22] The incorporation of metallocene fragments, usually cobaltocene or ferrocene, in such systems has been pursued essentially to sense or activate molecular level processes. Kaifer and coworkers reported a redox controlled dissociation of a self assembled dimer 23 whereas Beer took advantage of the receptor's electrochemical activity as a signal of a recognition event. 24,25 The latter has been pioneered in making metallocene appended calixarenes for analytic purposes or to investigate electrochemical interactions between multi redox architectures. 26 Among the most relevant examples, one find an hetero ditopic ferrocene receptors containing two ethyl ester calix [4]arene units bridged by a ferrocene amide moiety 27 or a tetraferrocenyl-calixarene in which metallocenes have been directly introduced on aniline-like fragments. 28 In this paper, we report the synthesis and the characterization of novel redox active calixarene-based receptors wherein ferrocene fragments have been introduced at the lower rim through anion-binding urea or amide connections. We performed a thorough 1 H NMR investigation on a series of calixarene-ferrocene receptors to estimate their hydrogen bondingdriven self-association properties and improve our understanding of the correlation between molecular structures and redox properties. Their anion binding properties have especially been investigated by electrochemical methods and NMR spectroscopy.

Experimental Section

Electrochemical analysis

All electrochemical experiments were carried out using a CH-Instrument potentiostat.

∆δ = (δ max /2P 0 )[(P 0 + S 0 + 1/K) -[(P 0 + S 0 + 1/K) 2 -4P 0 S 0 ] 1/2 ]
with ∆δ = δ x -δ initial ; δ max = δ final -δ initial ; P 0 : concentration of 14 ; S 0 : concentration of anion ; K: binding constant.

Synthesis

General Considerations:

Starting materials and solvents were obtained from commercial suppliers and used without further purification. TLC: silica gel 60 F254. NMR spectra were recorded on DRX 300 or DRX500 Brücker FT spectrometers. Abbreviation was used as: s (singlet), d (doublet), dd (divided doublet), t (triplet), q (quadruplet), m (multiplet) and l (large). Mass spectra were recorded by electrospay at the Mass Spectrometry centre.

The synthesis of compound 4, 32 11 24 and 7 50 was realized according to the literature procedure.

(3-bromopropyl)ferrocene 5:

3-Bromopropanoyl chloride (2.17 mL, 21. Triethyl phosphonoacetate (5.2 mL, 30 mmol) was added to a stirred suspension of sodium hydride (1.16 g, 31 mmol) in THF (50 mL) at 0 °C to give white foam. The mixture was allowed to warm to room temperature for 30 min then cooled in an ice bath and compound 7

(5 g, 23.3 mmol) was added as a solution in THF (50 mL). After 20 min the reaction mixture was allowed to warm to room temperature and stirred for 1 hour. Saturated aqueous ammonium chloride was then added to the mixture. Diethyl ether was added and the combined layers were washed with water, dried (MgSO 4 ) and the solvent removed under vacuum. Purification by column chromatography (silica gel dichloromethane) affords compound 8 in 98% yields (6.5 g).

Ferroceneproponic acid 9 :

A solution of 1 g (3.52 mmol) of ethyl ferrocenylacrylate 8 and 0.10 g (1 mmol) of palladiumon-charcoal in 50 ml of ethanol was degassed under argon for 30 min before saturated with hydrogen. The mixture was vigorously stirred for 24 h. The filtration and the concentration under reduced pressure gave 1 g (95%) of yellow crystals corresponding to ethyl 3ferrocenylpropanoate. 1 H NMR (300 MHz, CDCl 3 ): 4.31 (s, 9H, HFc), 2.45 (s, 4H, CH 2 ), 4.39 (q, 2H, J = 7 Hz, CH 2 ), 1.21 (t, 3H, J = 7 Hz, CH 3 ).

Potassium hydroxide (3.92 g, 70 mmol) in 10 mL of water was added to a solution of ethyl 3ferrocenylpropanoate (2.0 g, 7 mmol) in ethanol (60 mL). After 8 hours at reflux the reaction mixture was concentrated in vacuo then diluted with water and washed with diethyl ether. The was obtained in 83% yields (1.5 g). 1 H NMR (300 MHz, CDCl 3 ):11.03 (s br, 1H), 4.02-4.08 (m, 9H, HFc), 2.49-2.63 (m, 4H, CH 2 ). 13 C NMR (75 MHz, CDCl 3 ): 180.1, 87.6, 68.9, 68.3, 67.9, 67.0, 35.9, 25.0.

1,3-alternate tetraaminocalixarene 3

A solution of tetraphthalimidopropoxy-p-tert-butylcalix [4]arene (6.9 g, 5 mmol) in ethanol (100 ml) was refluxed with hydrazine (5 ml). After 8 h the solvent was removed under reduced pressure. The residue was dissolved in CHCl 3 , washed with water, dried (MgSO 4 )

and the solvent was evaporated. The formed powder was dissolved in chloroform and precipitated with hexane to give the pure compound in 90% yields (3.9 g). Compound 3:

Results and discussion

Synthesis

The 5,11,17,23-tetra(tert-butyl)-25,27-bis(3-aminopropoxy)-26,28-dihydroxycalix [4]arene 1, 29 5,11,17,23-tetra(tert-butyl)-25,26,27,26-tetra(3-aminopropoxy)-calix [4]arene in cone conformation 2 30 were prepared in two steps from p-tert-butylcalix [4]arene as described in literature. Calixarene 3 in 1,3-alternate conformation was obtained by reduction of the tetra(phthalimide) analogue preliminary prepared as described by Bhömer et al. 31 The ferrocene-acetic acid starting material 4 (scheme 1) was prepared as described in literature from the N,N-dimethylaminomethylferrocene in good yield (83%). 32 The methyliodide intermediate was treated with potassium cyanide to afford the ferrocenylacetonitrile which was then converted to the corresponding carboxylic acid 4.

The ferrocenebutyric acid 6 was previously prepared by reaction of the ferrocene with succinic anhydride followed by a Clemmensen reduction. 33 We report here an alternative approach from the ferrocene in four steps and 50% overall yield (scheme 2). After a Friedel-Craft acylation, the ketone was reduced to affording the (3-bromopropyl)-ferrocene 5. The last steps are identical to those detailed for the ferrocene-acetic acid 4, cyanide formation then hydrolysis. The ferrocene propanoic acid 9 34 was prepared from the ferrocene carboxaldehyde 7 via a Horner-Wardsworth-Emmons olefination in quantitative yield. The last steps are the double bond hydrogenation of 8 followed by a saponification of the ester function to afford 9 in 78 % yields. Finally, 11 was obtained in three steps as already described in the literature 28 from the ferrocene carboxaldehyde 7 previously transformed into the corresponding oxime 10 followed by the reductive amination and condensation with the 4-nitrophenyl chloroformate.

Metallocene redox-active fragments were introduced onto a calixarene framework using the the ferrocene acids 6 or 9 and HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate) as coupling reagent. This straightforward peptide-based strategy allowed us to isolate the bis(amido)-calixarenes 12 and 14 respectively in 68% and 71 % yield (scheme 3).

Ferrocene acids 4, 6 or 9 and aminocalixarene 1 were also linked via an urea connection through a Curtius rearrangement. 35 A variety of approaches towards calixarene-ureas have already been investigated. Most of them were based on the use of commercially available isocyanates [36][37][38] or isocyanate derivatives of calixarenes. 39,40 In this study, the targeted redox active receptors 14, 15 and 16 were prepared in one pot procedure without isolating the isocyanate intermediate by reacting the calixarene amine with ferrocene carboxylic acid in the presence of diphenylphosphoryl azide and diisopropylethylamine

The tetra-ureaferrocenyl derivative 17 was obtained in relatively low yield (15%) through a Curtius rearrangement between 9 and a tetra(amino)calix [4]arene 2 (scheme 4). However, this yield was improved up to nearly 90 % using the Paul Beer's strategy upon using 11 as ferrocene-urea precursor. 28 Finally, the peptide coupling from 3 with 6 in the presence of HATU gave the corresponding tetra(amidoferrocenyl) macrocycle 18 in 72% yield with a 1,3-alternate conformation. The structures of 12-18 were clearly established by NMR spectroscopy and electrospray mass spectrometry. The cone conformations adopted in solution by the calixarene frameworks of calixarenes 12-17 were deduced from the 13 C NMR experiments with the presence of resonance signals observed between 31 and 32 ppm corresponding to the ArCH 2 Ar fragments. 41 Moreover, these coumpounds are present in symmetrical cone conformation as proved by the low value of ∆δ between the signals of the two sets of aromatic protons (∆δ lower than 0.016 in DMSO-d 6 ) and tert-butyl groups (∆δ close to 0.05 ppm in DMSO-d 6 ) as already observed by Ungaro et al with peptidocali [4]arene bisureas. 42 The resonance signal located at 39.6 ppm which corresponds to the ArCH 2 Ar of 18 confirmed the 1,3-alternate conformation of the macrocycle.

NMR analysis

Information about the solvent accessibility of all NH protons in 12-18 and their level of engagement in H-bonding could be obtained from temperature coefficients. The relative change in chemical shifts recorded as temperature is changed by 1 K (∆δ/∆T) is indeed an accurate probe to estimate the level of hydrogen bonding in molecular systems. 42,43 The temperature dependence (∆δ/∆T) of the 1 H NMR chemical shifts attributed to the NH signals in compounds 12-18 was evaluated in DMSO-d 6 . A typical displacement observed with 15 between 298 and 353 K is depicted on Figure 1. In this polar and H-bond acceptor media, intermolecular hydrogen bonds and those established with solvent molecules are known to be readily cleaved by increasing temperature. We moreover known from literature that ∆δ/∆T values found below -4.0 ppb/K for amide protons unambiguously reveal the absence of intramolecular hydrogen bonding processes. 43 Temperature coefficients were measured for compounds 12-18 upon recording seven 1 H NMR spectra between 298 K and 353 K. These analyses were performed on 3.0 mM solutions to ensure the absence of significant molecular aggregation. The recorded and calculated results are summarized in Table 1.

Full NMR characterizations and exhaustive proton assignments were achieved, for all receptors, from 2).

Electrochemical analysis

Depending of their solubility, the electrochemical signatures of compounds 12-18 were investigated by cyclic voltammetry, RDE and square wave voltammetry on glassy carbon electrodes in dichloromethane, DMF, acetonitrile and DMSO using n-tetrabutylammonium perchlorate as electrolyte (TBAP, 0.1 M). All cyclic voltammograms featured one single wave corresponding to the simultaneous oxidation of both ferrocene fragments, highlighting the limited or non-existent communication between the iron centres. As proved by the experimental data reported in table 2, the ferrocene half wave potential significantly shifts towards less positive values when the number of atoms between the metallocene and urea fragments increases (see compounds 14 and 16 in CH 2 Cl 2 ). This significant displacement can be unambiguously attributed to the electron-withdrawing effect of the urea and amide fragments on the metallocene probe which logically strengthens as their linker shortens.

Whatever the macrocycle, the redox signatures did not reveal strong interactions between ferrocenes, as proved by the observation of one single wave on their cyclic voltammograms corresponding to the simultaneous oxidation of all iron centres. The weak "interaction level" between redox moieties could nevertheless subtly be appraised upon measuring and comparing the difference between oxidation and reduction peak potentials (∆E p ), assuming that hints of "communication" between redox centers should bring about inequivalence signalled by larger ∆E p values . The experimental values reported in As mentioned above, the observation of one single ferrocene centered oxidation wave results from the lack of communication between both centres. The expected intra-molecular association between urea fragments hence does not bring both metallocene in sufficient proximity to bring about significant electrochemically promoted electrostatic effects. It is now well established that the level of electrochemical connection in multi-redox architectures can be accurately estimated upon measuring discrepancies between experimental results and theoretical modeling. The electrochemical behaviour of molecules with multiple redox centres has indeed been the subject of numerous studies. 43,44 It has been especially demonstrated 45 that electron transfers to or from molecules containing identical, non interacting, electroactive centres should yield a single current-potential curve similar to that observed with single electroactive centre (∆E p = 58 mV at 25 °C) but with a magnitude determined by the total number of redox centres. When each centre is characterized by the same standard potential both redox processes are expected to appear as a single wave with peak potentials satisfying ∆E p = 58 mV. 46 Formal potentials E 1 ° and E 2 ° can not be determined in a straightforward manner but the easily measured difference between oxidation and reduction peak potentials irreversible oxidation process, at E ≥ 800 mV, centred on both phenol subunits of the calixarene skeleton. 47 A typical voltamogramm showing the ferrocene and phenol centred redox systems is depicted on figure 3. As discussed above, 14, 16 and 13 exhibit structural and chemical features fully suited to achieve an efficient complexation/sensing of anionic species. Urea and phenol groups are indeed among the most widely used binding fragments to trap or transport negatively charged species in biological systems. [START_REF] Sessler | Anion Receptor Chemistry[END_REF] The calixarene's lipophilic character and structurating effect associated to the anion binding properties of urea or amide and phenol are expedient complementary features defining 14, 16 and 13 as artificial anion receptors with great potential. In addition to these favourable chemical and structural aspects, both metallocene moieties can be easily activated, ie oxidized at low potential, to further improve the receptor anion binding ability through electrochemically triggered ion pairing effects between anionic targets and in situ mono or bis-positively charged receptors. 1

E m °,
(

Anion binding

The anion binding ability of these receptors could be quickly confirmed upon studying the effects of added anionic substrates on the chemical shifts of selected fragments. Addition of n-tetrabutylammonium dihydrogenphosphate to a CDCl 3 solution of 14 especially led to significant low field shifts of singlets attributed to the urea groups ( °, figure 4) and to a lesser extent to the phenol moieties (•, figure 4). Such perturbations could be observed with all of the studied receptors which were soluble in CDCl 3 (13,14 and 16) and clearly result from interactions between hydrogen-bond donors and electron rich anionic species. In agreement with previously reported studies, 44 these receptors exhibit however, at least in their reduced forms, relatively low binding constants with anionic species, especially in the case of 13 and 16 for which the binding constants could not be precisely determined.

On the other hand, titration of 14 conducted in CD 2 Cl 2 with dihydrogenphosphate anion, since the affinity for this anion is higher, yielded for instance curves which could be fitted according to a 1/1 calixarene/anion stoechiometry with an estimated binding constant of 36 ± 4 M -1 (figure 5). The 1:1 stoichiometry was confirmed by job plot with 1 H NMR experiment in CD 2 Cl 2 (figure 6). A symmetrical bell-shaped curve centered at a 0.5 molar fraction was observed, thus confirming the presence only of the 1:1 host/guest complex.

The ability of 13, 14, and 16 to complex anionic species and their potential in molecular electrochemical recognition were also investigated by cyclic voltammetry in dichloromethane electrolyte. The addition of increasing amounts n-tetrabutylammonium nitrate, sulphate, chloride or acetate to a 10 -3 M solution of these receptors did not induce measurable changes in the ferrocene centred oxidation wave. This lack of electrochemical response can be 13 attributed to weak associations between these anions and the phenol/amide/urea binding fragments and/or to limited interaction of the complexed anion with the neutral and/or oxidized metallocenes redox probes.

However, an effective electrochemical response was observed in presence of hydrogen phosphate anions (figure 7). Addition of the latter indeed resulted in a clear two wave behaviour wherein the intensity of the initial ferrocene based wave progressively decreases at the expense of a novel signal at a much less positive potential (figure 7A and7B). After adding one molar equivalent of anion, the intensity of both signals appeared comparable and full disappearance of the original wave was only observed when the anion/receptor ratio reached about two. Such evolution is fully compatible with a strong binding of one H 2 PO 4 anion per urea fragment leading, in fine, to a bis(hydrogenophosphate) complex. This 2/1 binding stoechiometry, while differing from that found from NMR experiments, can be easily explained upon considering that two positive charges are being electrochemically generated onto the calixarene receptors. These results are furthermore in agreement with previously reported studies conducted on similar receptors. [START_REF] Sessler | Anion Receptor Chemistry[END_REF] It needs to be stressed out that both waves remained fully reversible through the titration experiment and that the maximal potential shift (∆E 1/2 ) between the uncomplexed and complexed receptors electrochemical activities reached 155 mV. The addition of H 2 PO 4 -anions to a solution of 14 did moreover not significantly modify the shift between oxidation and reduction peak potentials (∆E, table 3). This observation is another clear indication that the "large" ∆E value measured in dichloromethane does not result from urea-driven intramolecular redox communication which would logically be disrupted upon adding hydrogen-phosphates.

Several interactions and effects can potentially be considered to account for the observed changes. The hydrogen bonding ability of urea and phenol groups is obviously of great importance but the influence of ferricinium and effect of complexation on ferrocene are also key elements to achieve such efficient molecular electrochemical sensing. A short linkage between urea and ferrocene was initially considered to be essential to optimize the transduction of any recognition process. In order to estimate the importance of the ureaphosphate bonds in the wave's displacement, which arises from an overall increase of electron density around the metallocene fragments, we studied the response of 16 and 13 in which ferrocene and binding fragments are fully disconnected, through a long alkyl chain, prohibiting any through bonds effects on the electrochemical recognition process. Although the most important perturbation is observed in the latter case, 16 and 13 still allow an effective sensing through important displacements of the ferrocene oxidation potential.

This result shows the utmost importance of ion pairing effects in electrochemical recognition process. When the ferrocene groups are far away from the hydrogen binding fragments, as in 16 and 13, the strong selective electrostatic interaction between hydrogen phosphate and the electrogenerated ferricinium species is almost enough to ensure an efficient transduction. This assumption is further demonstrated upon studying the electrochemical activity of simple ferrocene and TBA•H 2 PO 4 in dichloromethane. The initial reversible Fc/Fc + couple quickly evolve towards a broad irreversible system in presence of phosphate presumably resulting from the formation of poorly soluble ion pairs. The organic receptors used in this study have thus two main effects, besides introducing selectivity and affinity associated to the specific binding fragments, they also greatly help stabilizing and solubilizing the oxidized forms of the calixarene-anion complexes. The difference in ∆E 1/2 (ca. 40 mV, Table 3)) observed between 16•(H 2 PO 4 -) 2 and 13•(H 2 PO 4 -) 2 is tentatively attributed to distinct coordination modes setting the complexed anion closer to the redox probe in 16 than in 13. This shift is furthermore correlated to the number of atoms separating the ferrocene fragment from the amide hydrogen bond donor in both receptors.

Conclusion

In conclusion, we have described the synthesis of original calix [4]arenes wherein ferrocene fragments have been introduced at the lower rim through urea or amide connections. We

Fig. 1 :

 1 Fig. 1: 1 H NMR spectra of 15 in DMSOd 6 (500MHz) at 298 K. Enlargement showing the effect off temperature on both NH of the urea's chemical shifts.

Fig. 2 :Fig. 3 :Fig. 4 :

 234 Fig.2: expected H-bonding in compound 15

Fig. 5 : 4 -

 54 Fig. 5: ∆δNH i and ∆δNH ii as a function of [H 2 PO 4 -] for 14

Introduction

  Anion recognition and sensing are increasingly important research topics in supramolecular chemistry due to the entanglement of various anions in biological and environmental subjects.1,2 Chemists usually take advantage of a wide range of recognition fragments introduced on organic platforms to develop electrostatic, hydrophobic, π-π or hydrogen bonding interactions, and NMR shifts or photochemical signals as transduction signals.Calixarenes are particularly interesting frameworks to build on artificial anion binding architectures.3 Their dynamic properties, hydrophobic bowl-shape structures and well known chemical versatility are indeed fully suited to straightforwardly synthesize complex host molecules allowing multi-point recognitions of anionic targets. In the present study we the anion binding properties of novel artificial receptors

  acidified with HCl 1M and extracted with diethyl ether. The organic phase was dried over MgSO 4 , filtered and concentrated under vacuum. Ferroceneproponic acid 9

  and adheres to the Nernst equation independently of the oxidation state of any of the other centres in the molecule, it is possible to calculate the formal potentials corresponding to each pair of successive oxidation states of the multi-centres molecules. Considering fully non interacting centres, the theoretical shift between both ferrocene based formal oxidation potentials in 13, 14 and 16 (E 1 ° and E 2 °) should thus equal ∆E = E 1 °-E 2 ° = 35.6 mV, although

  especially assessed the presence and/or the lack of intramolecular hydrogen bonds by NMR spectroscopy. The anion binding properties of these artificial receptors have been revealed by NMR spectroscopy and thoroughly investigated by electrochemical methods. We especially assessed the importance of the urea-phosphate bonds in the observed electrochemical response upon studying receptors wherein ferrocene and binding fragments are intimately associated or fully disconnected through a long alkyl chain. Although similar selective phosphate sensing properties have been previously reported with intimately connected ferrocene-amide-calixarene receptors,26,[START_REF] Harvey | [END_REF] our experimental results clearly show the utmost importance of ion pairing effects in this electrochemical recognition process which accounts for more than 85 % of the transduction signal.

Fig. 7 :Fig. 6 :Page 32 of 32 URL

 7632 Fig. 7: (A) Cyclic (ν =100 mV.s -1 ) and (B) square wave voltammograms of 14 (5.10 -4 M) recorded in dichloromethane (0.1 M TBAP) upon adding increasing amounts of TBA•H 2 PO 4 (glassy carbon electrode, Ø = 3 mm).

Table 1 :

 1 ∆δ/∆T (ppb/K) 12-17 (DMSO-d 6 , 500MHz)

	1 )

11 Scheme 2. Synthesis of the ferrocenyl derivatives 6, 9 and 11 from ferrocene. 1 Scheme 3. Synthesis of redox-active bis-1,3-(amido)calixarenes 12,13 and bis-1,3-(urea)calixarenes 14,15 and 16.

Table 2 :

 2 Experimental E 1/2 and ∆Ep values (mV)*

		CH 2 Cl 2		DMF		CH 3 CN		DMSO
		E 1/2	∆Ep	E 1/2	∆Ep	E 1/2	∆Ep	E 1/2	∆Ep
	14	175	126	49	86	-	-	-	-
	15	-	-	-	-	-	-	-5	86
	16	139	106	25	78	39	70	-	-
	17	-	-	-	-	-	-	16	100
	12	-	-	-	-	56	68	-12	76
	13	153	98	36	84	42	77	-	-
	18	-	-	-	-	53	62	-	-

Table 3 .

 3 Electrochemical data (∆E, ∆E 1/2 ) measured by cyclic voltammetry before and after adding two molar equivalents of TBA•H 2 PO 4 to dichloromethane (0.1M TBAP) solutions of 14, 16 and 13 (5.10 -4 M, ν =100 mVs -1 , glassy carbon electrode Ø = 3 mm).

		13	13.(H 2 PO 4	-) 2	14	14.(H 2 PO 4	-) 2	16	16.(H 2 PO 4	-) 2
	∆E (mV)	92	108		98	100		92	98
	∆E 1/2 (mV)	94			155			135

  Potassium hydroxide (3.92 g, 70 mmol) in 10 mL of water was added to a solution of (3cyanopropyl)ferrocene (1.77 g, 7 mmol) in ethanol (60 mL). After 8 hours at reflux the reaction mixture was concentrated in vacuo then diluted with water and washed with diethyl ether. The aqueous phase was acidified with HCl 1M and extracted with diethyl ether. The organic phase was dried over MgSO 4 , filtered and concentrated under vacuum. The desired product was obtained in 84% yields (4.28 g) Mp = 85-86 °C 1 H NMR (300 MHz, CDCl 3 ): 4.12 (s, 5H, HFc), 4.07 (s, 4H, HFc), 2.37-2.42 (m, 4H, CH 2 ), 1.75-1.80 (m, 2H, CH 2 ).13 C NMR (75 MHz, CDCl 3 ): 179.9, 87.8, 68.5, 68.1, 67.2, 33.6, 28.8, 25.9. HRMS calculated for C 14 H 16 FeO 2 : 272.04997 found: 272.05044.

	Ferrocenebutyric acid 6:
	5 mmol) in CH 2 Cl 2 (8 mL) was added dropwise to a suspension of aluminium chloride (3 g, 22.5 mmol) in CH 2 Cl 2 (8 mL) at room temperature and stirred for 2 hours. After cooling (-10 °C), the homogenous solution was added to a solution of ferrocene (4 g, 21.5 mmol) in CH 2 Cl 2 (50 mL) cooled to 0 °C. The resulting purple solution was allowed to warm to room temperature and stirred for 16 hours. This solution was then diluted with CH 2 Cl 2 and poured over ice-H 2 O. The product was extracted with CH 2 Cl 2 and the organic phase was washed with saturated aqueous solution of NaHCO 3 , brine, dried over MgSO 4 , filtered and concentrated. After purification by flash chromatography (silica gel o r P e e r R e v i e w O n l CH 2 Cl 2 ), the desired product was obtained in 70% yield (4.85 g). F y
	4

1 H NMR (300 MHz, CDCl 3 ): 4.78 (t, 2H, J=1.88 Hz, HFc), 4.23 (s, 5H, HFc), 3.72 (t, 2H, J=6.60 Hz, CH 2 ), 3.29 (t, 2H, J=6.60 Hz, CH 2 ). 13 C NMR (75 MHz, CDCl 3 ): 200.6, 78.3, 72.6, 70.0, 69.3, 42.4, 26.1. Ethyl ferrocenylacrylate 8 51 :

  J=13 Hz, ArCH 2 Ar), 2.13-2.25 (m, 16H, CH 2 Fc, CH 2 CO,CH 2 CH 2 CH 2 ), 1.66-1.69 (m, 4H, CH 2 CH 2 CH 2 ), 1.32 (s, 18H, t-Bu), 0.99 (s, 18H, t-Bu). 13 C NMR (75 MHz, CDCl 3 ): 174.0, 150.4, 149.7, 147.7, 142.6, 132.7, 127.9, 126.0, 125.7, 88.9, 74.5, 69.0, 68.6; 67.7, 37.3, 36.5, 34.3, 34.2, 32.2, 32.0, 31.3, 30.5, KBr pellets cm -1 : 3370, 2959, 2869, 1651. HRMS calculated for C 76 H 97 Fe 2 N 4 O 6 : 1273.6112 found: 1273.612. Compound 16: 51%; Mp: 125-130 °C; 1 H NMR (300 MHz, CDCl 3 ): 7.73 (s, 2H, OH), 7.09 (s, 4H, ArH), 6.87 (s, 4H, ArH), 5.91 (s br, 2H, NH), 5.39 (s br, 2H, NH), 4.19 (d, 4H, J =

	4.05 (m, 4H, CH 2 O), 3.67-3.69 (m, 4H, CH 2 NH), 3.36 (d, 2H, 29.7, 29.2, 27.2. IR: KBr pellets cm -1 : 3299, 2954, 2866, 1643. HRMS calculated for 30.5. IR: 13.2 Hz, ArCH 2 Ar) 4.08 (s, 10H, HFc), 3.99-4.06 (m, 12H, HFc, CH 2 O), 3.64-3.70 (m, 4H,
	C 78 H 98 Fe 2 N 2 O 6 : 1270.61237 found: 1270.61244. CH 2 NH), 3.39 (d, J=13.2 Hz, ArCH 2 Ar), 3.19-3.26 (m, 4H, CH 2 NH), 2.27-2.31 (m, 4H,
	Compound 18: 72%; Mp = 139-143°C; 1 H NMR (300 MHz, CDCl 3 ): 6.99 (s, 8H, ArH), 6.03 CH 2 CH 2 Fc), 2.16-2.20 (m, 4H, CH 2 CH 2 CH 2 ), 1.61-1.67 (m, 4H, CH 2 CH 2 CH 2 ), 1.31 (s, 18H,
	(m, 4H, NH), 4.14 (m, 20H, HFc), 4.10 (m, 16H, HFc), 3.78-3.90 (m, 8H, ArCH 2 Ar), 3.13-t-Bu), 1.00 (s, 18H, t-Bu). 13 C NMR (75 MHz, CDCl 3 ): 159.5, 149.9, 149.6, 147.7, 142.9,
	F o r 3.30 (m, 8H), 2.90-3.03 (m, 8H), 2.08-2.45 (m, 8H), 2.07-2.21 (m, 8H), 1.72-1.88 (m, 8H, CH 2 CH 2 CH 2 ), 1.25-1.36 (m, 44H, CH 2 CH 2 CH 2 , t-Bu). 13 C NMR (75 MHz, CDCl 3 ): 173.2, 155.1, 144.5, 133.8, 126.4, 88.7, 68.9, 68.8, 68.5, 67.7, 39.7, 37.6, 36.45, 34.3, 32.1, 30.5, 30.1, 39.6, 27.5, 27.3. IR: KBr pellets cm -1 : 3299, 2951, 2866, 1643. HRMS calculated for 132.6, 128.0, 126.1, 125.7, 88.8, 75.8, 68.7, 68.2, 67.4, 40.4, 38.9, 34.3, 34.7, 32.2, 31.9, 31.7, F 31.3, 30.4, 27.0. IR: KBr pellets cm -1 : 3376, 2961, 2867, 1634. HRMS calculated for o r C 78 H 100 Fe 2 N 4 O 6 : 1300.63417 found: 1300.63419.
	Compound 12: 68% Mp = 223-225 °C; 1 H NMR (300 MHz, CDCl 3 ): 7.63 (s, 2H, OH), 7.10 (s, 4H, ArH), 6.85 (s, 4H, ArH), 3.96-4.17 (m, 26H, ArCH 2 Ar, HFc, CH 2 O), 3.62-3.67 (m, 4H, CH 2 NH), 3.34 (d, 4H, J=12.9 Hz, ArCH 2 Ar), 2.61-2.69 (m, 4H, CH 2 CO), 2.41-2.44 (m, P e e r R e v i e w O n l y P e e r R e v i e w C 112 H 141 Fe 4 N 4 O Bu), 0.98 (s, 18H, t-Bu). 13 C NMR (125 MHz, CDCl 3 ): 159.0, 149.7, 149.6, 147.8, 142.9, 132.6, 128.0, 126.1, 125.7, 86.1, 76.1, 68.9, 68.7, 68.3, 39.9, 39.2, 34.3, 34.2, 32.4, 31.9, 31.3, 30.1. IR: KBr pellets cm -1 : 3364, 2956, 2867, 1632. HRMS calculated for C 74 H 92 Fe 2 N 4 O 6 : 0, 154.1, 144.5, 134.2, 125.5, 88.2, 73.8, 69.2, 68.3, 68.09, 39.6, 39.3, 37.5, 34.3, 32.0, 31.8, O 31.2. IR: KBr pellets cm -1 : 3376, 2961, 2867, 1634. HRMS calculated for C 106 H 137 Fe 4 N 8 O 8 : n l y 1873.79516 found: 1873.79483.
	4H, CH 2 Fc), 2.05-2.14 (m, 4H, CH 2 CH 2 CH 2 ), 1.29 (s, 18H, t-Bu), 0.98 (s, 18H, t-Bu). 13 C 1244.57157 found: 1244.57163.
	NMR (75 MHz, CDCl 3 ): 173.5, 150.4, 149.7, 147.7, 142.7, 132.8, 128.0, 126.1, 125.7, 88.3, Compound 15: 51%; Mp = 159-162 °C; 1 H NMR (300 MHz, CDCl 3 ): 7.87 (s, 2H, OH), 7.09
	77.6, 74.4, 69.2, 68.7, 67.8, 38.7, 37.2, 34.4, 34.3, 32.3, 32.0, 31.4, 29.5, 26.2. IR: KBr pellets (s, 4H, ArH), 6.91 (s, 4H, ArH), 5.81 (s br, 2H, NH), 5.26 (s br, 2H, NH), 4.08-4.22 (m, 26H,
	cm -1 : 3333, 2954, 2869, 1635. Elem. Anal. Calculated C: 73.42, H: 7.62, N: 2.25 found: C: ArCH 2 Ar, HFc, CH 2 O), 3.64-3.70 (m, 4H, CH 2 NH), 3.20-3.34 (m, 8H, ArCH 2 Ar, CH 2 NH),
	6 7

1

H NMR (300 MHz, CDCl 3 ): 6.92 (s, 8H, ArH), 3.74 (s, 8H, ArCH 2 Ar), 3.

34

-3.42 (m, 8H, CH 2 O), 2.39-2.46 (m, 8H, CH 2 NH), 1.20-1.24 (m, 44H, CH 2 CH 2 CH 2, t-Bu). 13 C NMR (75 MHz, CDCl 3 ): 155.3, 144.0, 126.3, 69.1, 39.9, 39.6, 34.4, 33.8, 32.1. IR: KBr pellets cm -1 : 3369, 2958, 2901, 1482. HRMS calculated for C 56 H 84 N 4 O 4 : 877.6571 found: 877.6566.

Amide Synthesis:

HATU (1.2 mmol, 450 mg) and diisopropylethylamine (1.2 mmol, 0.2 mL) was added to a solution of ferrocenebutyric acid (1.2 mmol, 320 mg) in DMF (3 mL) and stirred for 15 min at 25 °C. This solution was then transferred to a suspension of 5,11,17,23-tetra(tert-butyl)-25,27-bis(3-aminopropoxy)-26,28-dihydroxycalix

[4]

arene (0.49 mmol, 370 mg) in DMF (5 mL) via a cannula. After 2 hours, the reaction mixture was concentrated and purified by flash chromatography (silica gel cyclohexane-ethyl acetate 6/4) to afford the compound 13 in 71% yields (443 mg). 73.19, H: 7.55, N: 2.13. Compound 13: Mp: 105-109 °C; 1 H NMR (300 MHz, CDCl 3 ): 7.56 (s, 2H, OH), 7.09 (s, 4H, ArH), 6.84 (s, 4H, ArH), 4.29-4.45 (m, 18H, HFc), 4.19 (d, 4H, J=13 Hz, ArCH 2 Ar), 4.01-8 : 1893.81416 found: 1893.81435. General procedure for Curtius reaction: diisopropylethylamine (9 mmol) and diphenylphosphoryl azide (4.4 mmol) were added to a solution ferrocenecarboxylic acid (2.2 mmol) was dissolved in toluene (15 mL). The reaction mixture was heated at 70 °C for 2 hours then the 5,11,17,23-tetra(tert-butyl)-25,27-bis(3aminopropoxy)-26,28-dihydroxycalix[4]arene (1 mmoles) was added. After 2 hours at 70°C, the reaction mixture was concentrated under vacuum and purified by flash chromatography (silica gel ethyl acetate-cyclohexane 6-4) to afford the desired product.

Compound 14: 55%; Mp: 143-146 °C; 1 H NMR (500 MHz, CDCl 3 ): 7.71 (s, 2H, OH), 7.04 (s, 4H, ArH), 6.83 (s, 4H, ArH), 5.94 (s br, 2H, NH), 5.40 (s br, 2H, NH), 4.11 (s, 10H, HFc), 4.02-4.08 (m, 16H, ArCH 2 Ar, HFc, FcCH 2 NH), 3.95-3.97 (m, 4H, CH 2 O), 3.59-3.63 (m, 4H, CH 2 NH), 3.30 (d, 2H, J=13 Hz, ArCH 2 Ar), 2.05-2.08 (m, 4H, CH 2 CH 2 CH 2 ), 1.29 (s, 18H, t-2.39-2.42 (m, 4H, CH 2 CH 2 Fc), 2.14-2.19 (m, 4H, CH 2 CH 2 CH 2 ), 1.30 (s, 18H, t-Bu), 1.03 (s, 18H, t-Bu). 13 C NMR (75 MHz, CDCl 3 ): 159.4, 149.9, 149.7, 148.1, 143.2, 132.9, 128.2, 126.2, 125.8, 77.7, 75.7, 69.6, 69.1, 68.7, 68.2, 41.8, 38.8, 34.5, 34.3, 32.4, 32.0, 31.4, 30.7, Tetra-ferrocene-urea functionalised calix[4]arene receptor 17. Tetra-aminocalix[4]arene derivative 2 (480 mg, 0.55 mmol) in CH 2 Cl 2 (20 ml) was added dropwise to a stirred solution of 11 (878 mg, 2.31 mmol), ethyldiisopropylamine (1.5 ml, excess) and DMAP (catalytic) in CH 2 Cl 2

(20 ml

) and the mixture was stirred for 24 h under argon. The reaction mixture was washed with sodium carbonate solution then the organic fraction was dried over magnesium sulfate, filtered, and the solvent removed under vacuum.

The residue was purified by column chromatography (silica gel ethyl acetate). The desired product was obtained in 89% yields (0.916 g). Mp >300 °C;

1

H NMR (500 MHz, DMSO-d 6 ): 6.78 (s, 8H, ArH), 6.22 (s br, 4H, NH), 6.02 (s br, 4H, NH), 4.27 (d, 8H, J = 12.3 Hz, ArCH 2 Ar), 4.13-4.16 (m, 28H, HFc), 4.05-4.07 (m, 12H, HFc), 3.95-3.97 (m, 8H, CH 2 NH), 3.76-3.82 (m, 4H, CH 2 O), 3.21-3.29 (m, 8H, CH 2 NH), 3.11 (d, 8H, J=13.2 Hz, ArCH 2 Ar),

2.08-2.11 (m, 8H, CH 2 CH 2 CH 2 ), 1.00 (s, 36H, t-Bu).

13

C NMR (75 MHz, CDCl 3 ): 159.

  1 H NMR and 2D-COSY spectra. From the low ∆δ/∆T values calculated for the bis-amide derivative 12, 13 and tetramide 18, we readily inferred the absence of interactions between amide arms. Surprisingly, both NHs of the urea linked ferrocenecalixarenes 14, 15 and 17 turned out to exhibit significantly different ∆δ/∆T values. between all four urea groups. In view of these results, ROESY analysis was carried on compound 15 and 17 to obtained more informations. Selective irradiation of each NH for compound 17 revealed a correlation between NH i (stands for NH's on the calixarene side) and O-CH 2 -CH 2 -CH 2 , NH b (stands for NH's on the ferrocene side) and between NH ii and NH i , CH 2 -HFc. Concerning compounds 14 and 15, the same type of correlation was observed for NH i than for compound 17. With a limit ∆δ/∆T value of -4.0 ppb concerning the NH ii of 14, a weak H-bonding interaction could be expected. However NH b correlated only with NH i . Based on these results which suppose that only NH i are engaged in a H-bonding, we assume that the urea was in trans configuration and/or the bottom part of the molecule was outside the cavity (figure

	hydrogen bonding network
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	Whereas NHs on the calixarene side (∆δ/∆T = -3.6 ppm/K) are clearly involved in
	intramolecular H-bonding, lower value (∆δ/∆T = -4.4 ppm/K) calculated for NH's on the
	10

ferrocene side suggest simple solvent effects. The most positive ∆δ/∆T values were found for the tetra urea-linked ferrocene calixarene 17. In contrast to what was found with 15, both NH's in 17 exhibit similar values strongly suggesting the existence of an intramolecular

Table 2

 2

	are interestingly

parameters might contribute to these deviation in electrochemical activities (number of redox centers, adsorbtion processes, kinetics…), the changes in ∆E p and ∆δ/∆T values, calculated in

  ∆E p ) gives reliable leads on the "communication level" between multiple redox centres. The experimental values reported in table 2 especially reveal the discrepancy in ∆E p values ranging from 98 to 126 mV in dichloromethane. Such deviation from theoretical prediction can be attributed to different factors, involving electron transfer kinetics, adsorption processes or ohmic drop contributions but also to non negligible interactions between electrogenerated ferricinium species. Such assumption especially relies on the fact that the largest ∆E p value was observed with 14, wherein intramolecular hydrogen bonds between urea groups expectedly lead to the shortest distance between both metallocene centres. This ∆Ep differences measured in apolar dichloromethane however turned out to be concentration dependent since a significantly smaller value was measured at 5 10 -4 M (table3). Such feature thus clearly suggested the absence of communication effect which should thus not be considered to account for the large ∆E value. All studied receptors exhibit a second

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  The interaction of the urea groups in 14 with H 2 PO 4 -species has indeed an unambiguous electronic, through bonds, effect on the ferrocene's final oxidation potential.

	highlight the beneficial effects of intimately connecting the recognition and signalling
	fragments.
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	14

Addition of increasing amounts of TBA•H 2 PO 4 to a dichloromethane solution of the free receptors 16 and 13 surprisingly led to similar "two wave" responses only distinguished by the half waves potential shifts amplitudes (∆E 1/2 , table 3). The ∆E 1/2 values reported in table 3 indeed clearly

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