Iwona Szymańska

Eddy Dolusic

Wim Dehaen

Wouter Maes

Takashi Ito
email: ito@ksu.edu

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

Determination of Interaction Strength between Corrole and Phenol Derivatives in Aqueous Media Using Atomic Force Microscopy

Keywords: atomic force microscopy, molecular recognition, corrole, phenol derivatives, aqueous media atomic force microscopy, molecular recognition, corrole, phenol derivatives, aqueous media

Atomic force microscopy (AFM) was used to measure single interaction forces between corrole (host) and phenol derivatives (guests) in aqueous media. A gold tip was modified with thiol derivatives of corrole via the Au-S covalent bond. Such a tip was used to measure adhesion forces with a planar gold substrate modified with thiol-derivatives of phenol and ortho-nitrophenol in aqueous solutions. The mean force between the corrole and orthonitrophenol was higher than that between corrole and phenol, probably reflecting stronger hydrogen bond interaction in the former complex. In the presence of supporting electrolyte (0.1 M K 2 SO 4 ), the mean force increased, suggesting that electrostatic and π-π interactions play an essential role in the adhesion force. In addition, adhesion force measured at pH 6.0 was larger than that at pH 10, reflecting the electrostatic repulsion at the higher pH. These behaviors are consistent with the potentiometric responses of a liquid membrane based on corrole to phenolic compounds. Also, the values of forces for interaction between corrolephenol derivatives showed the same tendency as energy calculated for these complexes. The Poisson method was used for the calculation of the single force of the chemical bond between the corrole host and the phenolic guests.

INTRODUCTION

The exploring of the mechanism of the host -guest recognition at the molecular level is a valuable source of inspiration for researchers dealing with sensors which are working by mimicking physiological processes occurring with outstanding sensitivity and selectivity. The which deals with the objects as small as 1 -100 nm [1][2]. Images of surfaces at such an extremely small scale, down to resolving individual atoms or molecules, can be obtained by newly developed technologies, namely scanning tunneling microscopy (STM) and atomic force microscopy (AFM) [3 -9]. The introduction of chemically tailored STM tips by Umezawa and co-workers dramatically improved the sensitivity of visualization of molecular recognition processes at the membrane/solid interfaces [10][11][12][13][14][15][16][17][18]. The chemical modification of STM tips [10][11][12][13][14][15][16][17][18] as well as AFM tips [19,20] increases the chemical affinity between the imaging tip and the substrates through formation of hydrogen bonds, metal -coordinationbonds and charge -transfer interactions. This remarkable cutting-edge technique has opened the possibility to explore the host -guest interactions at the molecular level between nucleobases [18], drug -enzyme [21], antigen -antibody [22,23], and other biomolecules [24,25], just to name a few.

The main objective of the research presented is showing the applicability of the AFM method together with chemically tailored tips for the exploring of the molecular recognition processes between corroles and phenol derivatives occurring at the solid /aqueous interface.

It has been already discovered by the potentiometric method that liquid membrane electrodes incorporating macrocyclic polyamines [26][27][28][29], calix [4]pyrroles [30][31][32][33] or corroles [34][35] upon stimulation with undissociated phenol derivatives generated potentiometric responses.

The mechanism of this unusual phenomenon, generation of potentiometric signals by neutral molecules, relies on the transport of protons from the interface to the aqueous layer adjacent to the organic phase.

The host molecule being the subject of this study is corrole. This molecule belongs to the tetrapyrrolic macrocyclic compounds with 18 π electrons. Corroles contain one direct linkage between two adjacent pyrrole rings. They display unusually high N-H acidity relative to other related macrocycles [36]. In this paper we present a study on the interaction forces between corrole and phenol derivatives immobilized through Au-S bonds on a gold AFM tip and a gold substrate surface, respectively. The influence of the pH and the composition of aqueous solution on the strength of these interactions was also explored. These experiments have addressed to determination of type of forces between corrole host and phenolic guests, measured in aqueous media.

EXPERIMENTAL

Materials and Reagents

The gold tip and substrates modification together with the chemical structures of the corrole and phenol derivatives used in the present study are illustrated in Scheme 1. Phenol, and ortho -nitrophenol possessing SH group were received from ProChimia Surfaces (Sopot, Poland). Planar gold substrates (Au.1000.ALSI) were purchased from Platypus Technologies (Madison, USA). AFM cantilevers, commercially available (contact mode, resonant frequency 13 kHz, force constant 0.2 N/m) were obtained from Budgetsensors (Sofia Bulgaria). All aqueous solutions were prepared with deionized water purified with a Milli-Q water system. All the chemicals were analytical grade, used as received.

Synthesis of corrole

Corrole was synthesized at the Department of Chemistry (University of Leuven) using method [37][38][39] as described below:

4-(10-Undecenoxy)benzaldehyde

A mixture of 7.030 g (28.64 mmol) of 11-bromo-1-undecene, 4.892 g (39.66 mmol) of p-hydroxybenzaldehyde and 8.328 g (60.26 mmol) of potassium carbonate was heated at reflux in 105 ml of acetone (p.a.) for 15 h. Upon cooling, the reaction mixture was filtered, evaporated to dryness and the residue was taken into dichloromethane. The solution was washed three times with aqueous NaOH, then three times with water and dried over MgSO 4 .

F

4-(11-Thioacetoxyundecyl)benzaldehyde

4-(10-Undecenoxy)benzaldehyde (7.450 g; 27.15 mmol), thioacetic acid (4 ml; 56.23 mmol) and azo-bis-isobutyronitrile (229 mg; 1.37 mmol) were dissolved in 90 ml of toluene (p.a.). The mixture was degassed with a stream of argon and then refluxed for 6.5 h. The reaction was quenched with 5% aqueous NaHCO 3 (400 ml) and extracted three times with ethyl acetate. The combined organic layers were washed with 5% aqueous NaHCO 3 , then brine and dried over MgSO 4 . Upon filtration and removal of the solvent under reduced pressure, the yellow solid residue was chromatographed on silica eluting with a gradient of 5:1 to 1:1 light petroleum ether / ethyl acetate. The crude product obtained was recrystallized from methanol to afford 4.086 g (43%) of a white powder. δ H (300 MHz) 9

Meso-5,15-bis(2,6-dichlorophenyl)-10-(4-(11-mercapto-1-dodecyloxy) phenyl)corrole

Acetyl protected corrole (130 mg; 0.14 mmol) was dissolved in 7.5 ml of THF and 3 ml of methanol. The flask was placed in an ice bath and 1 ml of a

Formation of SAMs on gold substrates and gold tips

Before the modification the substrates were sonicated for 10 minutes in ethanol, dried with argon and annealed in a hydrogen flame. Next, the gold substrates were immersed in 10 mM ethanolic solutions of derivatives of phenol (Ph) and nitrophenol (PhNO 2 ), for 20 hours.

Next, the substrates were rinsed with ethanol and water and dried in air. At least, 3 repetitions of the substrate modification samples were done.

The gold tips were washed with ethanol, pretreated in an UV/ozone chamber (Novascan Technologies, Ames, USA) for 30 min., and then immersed in 0.5 mM corrole (COR) chloroform solution for 24 hour. Thus, 5 tips were modified and used for the experiments.

AFM measurements

AFM measurements were carried out with a Molecular Imaging PicoSPM (Agilent Technologies, Tempe, USA). Substrates: Ph or PhNO 2 and cantilever COR/tip were loaded in a liquid cell, which was allowed to reach thermal equilibrium before starting the AFM operation. Adhesion forces were determined from force-displacement curves at room temperature (ca. 20 °C) with the substrate and tip immersed in, milliQ water, an aqueous solution containing 0.1 M K 2 SO 4 at pH 6.0 or pH 10.0, with using the scanning rate of 1.17 lines/s. For each combination of tip and substrates, and appropriate pH of solution, at least 300 force-displacement curves were measured at different positions of each substrate -tip combination.

RESULTS AND DISCUSSION

Strength of adhesion forces between the corrole host and the phenolic guests measured in water by AFM

The experiments to study the interactions between the corrole host and the phenolic guests were carried out by using modified gold substrates and tips. The modification procedures are illustrated in Scheme 1.

First, interactions between a COR/tip and Ph-or PhNO 2 -substrates were assessed in water at room temperature. The tip and substrate were mounted in liquid cell and the forcedisplacement curves were monitored. Figure 1 shows a typical force-displacement curve observed between a PhNO 2 -substrate and a COR/tip. In many measurements, an adhesion force as shown in the Figure 1 was observed in the retracting part of the curve. In this paper, the host-guest interactions in water will be verified based on adhesion force, since it has often been used to discuss various intermolecular interactions [40]. Adhesion forces obtained from multiple measurements using a single tip on different locations of a sample were summarized in a histogram, which show the probability of a given value of adhesion forces. Figure 2A and Figure 3A show histograms observed with a COR/tip on a PhNO2-substrate and a Ph- 1).

F
In order to assess the contribution of nonspecific interactions to the aforementioned adhesion force data, force-distance curves on Ph-or PhNO 2 -substrates were measured using unmodified gold tip. Figure 4A and 4B shows histograms obtained from adhesion force data for a single tip. The adhesion forces were 1.0 ± 0.2 nN and 0.7 ± 0.2 nN, for Ph-and PhNO 2substrates, respectively. These values were smaller than those obtained with COR-tip. In addition, bare gold tips gave slightly larger adhesion force on Ph/substrates whereas COR-tips gave larger force on PhNO 2 /substrate. These results suggest that the difference in adhesion force measured using COR-tips reflects from the host-guest interactions.

The adhesion force reported above reflects not only the host-guest interactions but also the contact area originating from the variation in tip size [START_REF] Israelachvili | Intermolecular and Surface Forces London: Academic[END_REF]. The pull -off features in the retracting part of the force -distance curve can be used to calculate also the strength of a single chemical bond [START_REF] Han | [END_REF][43][44][45][46], which is not affected by the variation of the contact area. It has already been reported that AFM contact forces obeyed a Poisson distribution. Thus, the ratio of their variance to the mean gives the force of a single chemical bond between the tip and the From the slope of the linear relationship between the mean force and its variance, the single molecule bond force can be calculated (Figure 2B, Figure 3B). The calculated single molecule bond forces were 16 ± 3 pN for the Ph-and 75 ± 11 pN for the PhNO 2 -substrate, respectively (Table 2). Thus, the strength of the individual bond force between corrole and ortho-nitrophenol was almost five times higher in comparison with the force between corrole and phenol. These values are in a similar range as reported by Beebe Jr. et al. [START_REF] Han | [END_REF] and Hadziioannou et al. [47]. Their reports deal with a study of the hydrogen bonding, van der Waals and Coulombic interactions between ω -functional n -alkanethiol compounds with a variety of functional groups (CH 3 , OH, NH 2 , COOH and CONH 2 ) which were used for modification of scanning force microscopy probes and substrates.

pH dependence on the strength of the adhesion forces between the corrole host and the phenolic guests measured in 0.1 M K 2 SO 4 by the AFM method

In order to explore the influence of the pH and the electrolyte on the strength of interactions between the host and guests studied, subsequent experiments were done in the presence of 0.1 M K 2 SO 4 at two pH values: 6.0 and 10.0.

At pH 6.0, the shape of force-distance curves was similar to that observed in water (Figure 1). At that pH, corrole exists as a mixture of electrically neutral and deprotonated molecules on the surface of the gold tip [34]. On the other hand, the phenol and orthonitrophenol derivatives exist as the undissociated compound [31,32].

The histograms for the adhesion forces obtained for COR/tip and Ph-or PhNO 2substrate measured at pH 6.0 and their corresponding plots of mean force vs variance are given in Figures 5 and6. The average of adhesion force of COR-tips with PhNO 2 /substrates (5.0 ± 0.8 nN; Figure 5A and Table 1) is twice larger than that with Ph/substrates (2.2 ± 0.6 nN; Figure 5A and Table 1). The single bond force between corrole and PhNO 2 was also larger (140.5 ± 33.0 pN; Figure 5B and Table 2) than that between corrole and Ph (59.3 ± 10 pN; Figure 6B and Table 2). This result is again consistent with the stronger interactions between the corrole molecules and ortho-nitrophenol in comparison with phenol.

Adhesion force between the tip modified with corrole and substrates was not observed at pH 10 (Figure 7). Recorded force -distance curves displayed the typical shape for the repulsive forces between the functionalized tip and surface [40]. This lack of adhesion force is mainly caused by electrostatic repulsion between the tip and substrate. At

Computer simulation on the complexation between corrole and phenols

In addition, the difference in adhesion force observed for PhNO 2 -and Ph-substrate would originate from hydrogen bond interactions with corrole. The creation of supramolecular assemblies is controlled by N-H⋅⋅⋅X and O-H⋅⋅⋅X (X = O, N) hydrogen bonds.

The majority of recent publications are connected with molecular complexes of phenolic substrates with a variety of receptors [50,51]. These reports show that the supramolecular assembly is controlled by strong and directional O-H⋅⋅⋅N hydrogen bonds. The phenolic compounds can create a supramolecular complex with corrole [33,34]. The interaction involves the creation of a hydrogen bond between the NH group of the macrocyclic cavity of the corrole and the OH group of the phenol derivative. The oxygen from the phenolic host is an acceptor of hydrogen, while the NH group is a donor of hydrogen.

The significance of the hydrogen bond interactions in the host-guest supramolecular complex was verified using AM1 geometry procedure (MOPAC 2002 Version 2.5.3, J. J. P. Stewart, Fujitsu Limited, Tokyo, Japan). The calculation of a number of different conformations of the corrole and guest compounds was done using the CaChe Workspace programme in vacuum (CaChe Worksystem Pro Version 7.5.0.85). In Scheme 2, a model of the complex formation between corrole and ortho -nitrophenol derivatives is presented, as optimized by the AM1 geometry procedure. However, the calculation showed that the binding energy of ortho -nitrophenol is in a range of -5.8 kcal/mol. On the other hand, the phenol binding strength is a little less energetically favorable (-3.5 kcal/mol). In order to correlate the energy binding and force of the complex formation of corrole with phenol and orthonitrophenol, the procedure described by Reinhoudt was applied [52]. In this procedure, the dependence of pull-off forces vs. energy was achieved for the spherical and cylindrical integration approaches, according the following equations:

F = 32.60x 39 . 2 + ∆ -G or F = 31.76x 03 . 1 + ∆ -G
, respectively (with -∆G in kcal/mol and F in pN). Taking into account cylindrical integration approaches, the calculated values of forces were 83.0 pN and 67.6 pN, for PhNO 2 and Ph, respectively. The difference between predicted and experimental values is therefore as follows: 83.0/140.5 and 67.6/59.3 pN, respectively, as compared for the values obtained in 0.1 K 2 SO 4 , pH 6.0. The differences between calculated and experimental data could be caused by possible contribution of other kind of forces (e.g. π-π interactions or hydratation of water molecules to the complex). However, the results of calculation show the similar tendency as experimental data described in this paper.

The results obtained using AFM together with a chemically modified tip also correlate well with those obtained potentiometricaly, which concern the studies of corroles as the selective receptors for phenol derivatives existing as the electrically neutral analytes [33][34][35].

The presence of a nitro group on the benzene ring increases the acidity of the phenolic OH group, and as a consequence, a stronger potentiometric signal was observed. The significant part of the recognition process is the formation of weak hydrogen bonds between the electron pairs of the phenolic OH group and the NH units belonging to the corrole. This phenomenon was proved here, on the molecular level, by AFM method together with a chemically modified tip and substrates.

CONCLUSIONS

We presented a study on the strength of the interactions between a corrole host and phenolic guests using the AFM method together with gold substrates and tips modified with SAMs thorough Au-S covalent bonds. The specific intermolecular forces between corrole covalently immobilized on the gold AFM tip and derivatives of phenol, also attached via a sulfur -gold bond to the surface of substrates was observed by means of atomic force microscopy. The dominant nature in the selectivity of interactions between corrole host and phenolic guest are hydrogen bonds. Also, hydrophobic and π-π interactions between phenol and pyrrole rings are involved. The strength of the single adhesion forces between corrole host and phenolic guest were determined by the Poissson method analysis. The sequence of the strength of the single bond force obtained with the AFM method correlate well with the calculated values of energy and the selectivity of potentiometric signals obtained by liquid membrane electrodes incorporating corrole after stimulation with neutral phenolic guests [33][34][35]. The results obtained confirm the possibility to apply the atomic force microscopy for the observation at the molecular level of the creation of supramolecular complexes between nitrogen containing macrocycles and uncharged phenol derivatives. Notably, the nitrophenol isomers were recently identified as vasodilators which can mimic hormones, and can be considered as endocrine disrupting chemicals possessing estrogenic activity [53]. Thus, looking for the compounds which could specifically bind these molecules is very important from the theoretical and practical point of view. On the other hand, taking into account the interesting features of corrole which might be protonated and associate with a low pH cancer

  source of the intensive development of the above study is a new research field, nanoscience,

2 and

 2 SH), ~ -1.9 (br. s, 3H, NH); δ C 158.8 (CO), 142.1 (9-C and 11-C of corrole), 139.8 (6-C and 14-C of corrole), 138.5, 137.4, 135.5, 134.6, 134.0, 130.7, 130.3, 128.0 (3-C and 5-C of 5,15-dichlorophenyl), 127.1, 125.7 (8-C and 12-C of corrole), 120.8 (A and D ring -pyrrole C), 116.2 (A and D ring -pyrrole C), 113.2 (A and D ring -pyrrole C), 111.7 (5-C and 15-C of corrole), 108.9 (10-C of corrole); ESI-MS 865 (MH + ).

  substrate. The Poisson method requires relatively few measurements. By plotting a linear regression curve of the variance vs. the mean force taken from several sets of AFM measurements done at different locations on the surface and different tips, the magnitude of the individual bond force can be estimated. The lack of assumptions about the tip radius is another important advantage of this method. Indeed, the Poisson statistical analysis method has been widely used for the calculation of individual bond forces between the AFM tip and the substrate, both functionalized[START_REF] Han | [END_REF][43][44][45][46]. Thus, the Poisson method was used in the present study for the evaluation of the strength of single molecule bond forces between corrole and or ortho -phenol derivatives. Corroles due to the steric hindrance create less packed monolayers on the surface. Moreover, because of presence of short ethylene chains, the phenol derivatives SAMs are less ordered. These factors cause that the density of the host and guest on the surface is rather low. Thus, the Poisson method is suitable for the systems studied.

1 -

 1 pH 10.0, both corrole molecules and phenol and ortho-nitrophenol derivatives exist in the negatively charged forms. The pK a s of ortho-nitrophenol and phenol are 7.21 and 10.0, respectively. Thus, at pH 10.0, both the host and guests molecules obtain a negative charge. Similar phenomena, namely the switching in binding forces in relation to a change of the pH, were observed in several studies. The deprotonation effect of the ammonium group on the surface of the substrate under basic condition caused a decrease in the interaction between ammonium ion complexes with crown ethers moiety as presented by Kimura et al.[48]. A decrease in adhesion force with increased H + concentration for amino -or 2 -imidazolinyl terminated SAMs is a result of the repulsion between the positively charged tip and the substrate surfaces as reported by Umezawa et al.[49]. In the present study, we have demonstrated that the individual interaction forces between corrole and phenol as well as derivatives attached to the surface of a gold tip and gold substrates depend on the protonation/deprotonation conditions of both the host and guests. By changing the pH of the electrolyte solutions, switching in the binding force is possible. As summarized in Table2, the strongest single bond force was observed between the corrole and the orthonitrophenol derivatives measured at pH 6.0. The presence of electrolyte, 0.1 M K 2 SO 4 , increased the strength of the interactions, ca four times for corrole -phenol, and two times for corrole -ortho nitrophenol, in comparison to the values obtained in pure water. The larger adhesion force in the presence of 0.1 M K 2 SO 4 suggests the involvement of hydrophobic interactions (e.g., π-π interactions) in the supramolecular complex formed between the corrole and phenols.

Scheme 1 . 2 .

 12 Scheme 1.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

Figure 7 .

 7 Figure 7.

Figure 2 .Figure 3 .

 23 Figure 2.

Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 4567 Figure 4.

  Upon filtration and removal of CH 2 Cl 2 by distillation under reduced pressure, product 152 was purified by column chromatography on silica. Eluting with 1:1 dichloromethane / light petroleum ether afforded 7.450 g (95%) of the title product. δ H (300 MHz) 9.88 (s, 1H,

	aldehyde H), 7.82 (d, J 8.1, 2H, aryl H), 6.99 (d, J 8.1, 2H, aryl H), 5.81 (m, 1H, undecenyl
	10-H), 4.97 (m, 2H, undecenyl 11-H) 4.04 (t, J 6.6, 2H, undecenyl 1-H), 2.04 (q, J 7.0, 2H,
	undecenyl H), 1.81 (quintet, J 7.0, 2H, undecenyl 2-H), 1.46 -1.31 (m, 12H, undecenyl H).
	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Meso-5,15-bis(2,6-dichlorophenyl)-10-(4-(11-thioacetoxy-1-dodecyloxy) phenyl)corrole

	The solution was bubbled with Ar for 15 min. Ten l of TFA (0.13 mmol) was added and the
	mixture was stirred under an argon atmosphere for ~48 h. The ice bath was removed and 935
	mg (3.77 mmol) of p-chloranil was added. After additional 1 h at room temperature, the
	mixture was evaporated with silica and chromatographed twice in a 1.5:1 mixture of
	dichloromethane and n-heptane to afford 452 mg (29%) of corrole. δ H 9.00 (d, J 4.1, 2H, 2-H
	and 18-H of corrole), 8.62 (d, J 4.7, 2H, 8-H and 12-H of corrole), 8.53 (d, J 4.7, 2H, 7-H and
	F 13-H of corrole), 8.42 (d, J 4.1, 2H, 3-H and 17-H of corrole), 8.08 (d, J 8.5, 2H, aryl o-H), o r 7.78 (d, J 7.7, 4H, dichlorophenyl m-H), 7.66 (dd, J 1 7.7, J 2 8.7, 2H, dichlorophenyl p-H),
	7.27 (d partially overlapped with CHCl 3 , J 8.5, 2H, aryl m-H), 4.24 (t, J 6.5, 2H, OCH 2 ), 2.89 P e (t, J 7.3, 2H, SCH 2 ), 2.32 (s, 3H, CH 3 ), 1.98 (quintet, J 7.4, 2H, -CH 2 ), 1.62 -1.36 (m, 16H,
	8CH 2 ), -2.30 (br. s, 3H, NH); δ C 196.0, 158.9, 138.6 (quaternary C), 137.4 (quaternary C), e r 135.5 (CH), 134.0 (quaternary C), 130.3 (dichlorophenyl p-C), 128.0 (dichlorophenyl m-C),
	R 127.2 (pyrrole CH), 125.7 (pyrrole CH), 120.8 (C-3 and C-17 of corrole), 116.2 (C-2 and C-
	e 18 of corrole), 113.2 (CH), 111.7 (quaternary C), 108.8 (quaternary C), 68.3, 30.6, 29.6, 29.5, v e i 29.1, 28.8, 26.2; UV-Vis 410.1 (1.35 . 10 5 ), 566.1 (0.22 . 10 5 ); ESI-MS 907 (MH + ).
			w		.88 (s, 1H,
	aldehyde H), 7.83 (d, J 8.6, 2H, aryl H), 6.99 (d, J 8.6, 2H, aryl H), 4.04 (t, J 6.6, 2H, undecenyl 1-H), 2.86 (t, J 7.3, 2H, undecenyl 11-H), 2.32 (s, 3H, CH 3 ), 1.81 (quintet, J 7.3, O n l 2H, undecenyl 2-H), 1.46 -1.28 (m, 16H, 8CH 2 ). y
	2,6-Dichlorophenyldipyrromethane	(1.522	g;	5.23	mmol)	and	4-(11-
	thioacetoxyundecyl)benzaldehyde (606 mg; 1.73 mmol) were dissolved in 146 ml of
	dichloromethane. The reaction flask was wrapped in aluminium foil and placed in an ice bath.

  substrate, respectively. The average of adhesion force, measured using multiple COR/tips, was slightly larger for the PhNO 2 -substrates (2.1 ± 0.5 nN) in comparison with the Phsubstrate (1.7 ± 0.3 nN) (Table

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

  cell, these molecules can be used as sensors in biological systems and therefore there is still need for basic research connected with analogous systems.

	[48] Kado, S.; Yamada, K.; Kimura, K. Langmuir 2004, 20, 3259.
	[49] Ito, T.; Citterio, D.; Buhlmann, P.; Umezawa, Y. Langmuir 1999, 15, 2788.
	[50] Vishweshwar, P.; Nangia, A.; Lynch V. M CrystEngComm 2003, 5, 164.
	[51] Ghosh, K.; Datta, M.; Frohlich, R.; Ganguly, N. C. J. Mol. Struct. 2005, 737, 201.
	[52] Auletta, T.; de Jong, M. R., Mulder, A.; van Veggel, F. C. J. M.; Huskens, J., Reinhoudt,
	D. N.; Zou, S.; Zapotoczny, S.; Schonherr, H.; Vansco, G. J.; Kuipers, L. J. Am. Chem. Soc.
	F o r F o r F o r F 2004, 126, 1577. o r [53] Furuta, Ch.; Suzuki, A. K.; Taneda, S.; Kamata, K.; Hayasi, H.; Mori, Y.; Li, Ch.;
	P P P P Watanabe, G.; Taya, K. Biol. Reprod. 2004, 70, 1527.
		e e e e
	List of Figures and Tables:	e r e r e r r e
		R R R R
		e e e e
		v i e v i e v i e v i e
		w w w w
		O n l O n l O n l O n l
		y y y y

Table 1 .

 1 Adhesion forces measured with unmodified and corrole-modified tips and gold substrates modified with Ph or PhNO 2 in different aqueous solutions. Single bond forces measured with corrole-modified tips and Ph and PhNO 2 compounds immobilized on gold substrates in different aqueous solutions. Structure of the complex between corrole with an ortho -nitrophenol derivative optimized by AM1 geometry (CaChe Worksysem Pro Version 7.5.0.85).

	Table 2.

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Schematic representation of corrole -SH molecules (COR) immobilized on the surface of a gold AFM tip (A) and Ph (B), PhNO 2 (C) compounds immobilized on gold substrates. Page 22 of 32 URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Typical force curve (line "B" -approaching, b line "A" -retracting) observed for PhNO 2 -substrates using COR/tip made in water. Page 24 of 32 URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu

Table 1 .

 1 Adhesion forces measured with unmodified and corrole-modified tips and gold substrates modified with Ph or PhNO 2 in different aqueous solutions.

	Page 30 of 32

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

Table 2 .

 2 Single bond forces measured with corrole-modified tips and Ph and PhNO 2 compounds immobilized on gold substrates in different aqueous solutions.

	Page 32 of 32

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

Acknowledgements

We gratefully acknowledge financial support from the Kościuszko Foundation (to I. S.), EU grant COST D31/0021/05 (to I. S., H. R., E. D., W. D., W. M), Polish Ministry of Science and Higher Education 19/COS/2006/3 (to I. S. and H. R.), the F.W.O, the University of Leuven and the Ministerie voor Wetenschapsbeleid (to E. D., W. D., W. M.) and Kansas State University (to T.I.).

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


Elias Bou Maroun

Agnès Hagège

Christian Basset

Eric Quéméneur

Claude Vidaud

Zouhair Asfari
email: asfariz@ecpm.u-strasbg.fr

Synthesis of phosphorylated calix[4]arene derivatives for the design of solid phases immobilizing uranyl cations

Keywords: Phosphorous-Calix[4]arenes, Uranyl immobilization, Solid supports, Anchoring arms uranyl-affine Phosphorous-Calix[4]arenes, Uranyl cations immobilization, Solid supports, Anchoring arms

Synthesis of phosphorylated calix[4]arene derivatives

for the design of solid phases immobilizing uranyl cations.

Introduction

Uranium, especially under its uranyl ion form (UO 2 2+ ), is widespread in the environment, naturally occurring in various minerals but also resulting from both nuclear civil and military uses. Regarding to the complexity of biological matrices, studying its speciation in vivo still requires innovative tools. Thus, the design and synthesis of macrocyclic ligands which could preorganize uranyl chelating functions is of primary importance in the development of such tools.

Among them, calixarenes represent an interesting class of macrocycles. On the one hand, they might adopt pseudoplanar configuration. However, in contrast to homooxacalix [4]arenes and tetrathiacalix [4]arenes [1][2][3], the crystal structure of the complex between the simple calix [4]arene and UO 2 2+ evidenced that the 1,3-oxygen atom distance is far from the ideal distance required for an internal complex [3]. On the other hand, calix [4]arenes can adopt an 1,3-alternate conformation where the uranyl cation binding sites can be remote from the anchoring arm designed for a covalent bond to a polymer support.

Calix [4]arenes functionalized by various complexing groups were then used as extractants of uranyl ion, i.e. carboxylic acid [4][5][6][7][8], carboxylic acid-amide [9,10], ester [11], hydroxamate [12] or semicarbazone functions [13]. Phosphoryl groups have also been proved to be suitable for or acid organo-phosphorus extractants [14][15][16][17][18][19][20][21]. A recent work of Taran et al. reported that bisphosphonates were powerful uranyl ligands [22]. Several papers described the synthesis of phosphonatocalix [4]arenes [23][24][25][START_REF] Ozegowski | Sulfur and Silicon and Their Related Elements[END_REF][START_REF] Gloede | Phosphorus, Sulfur and Silicon and Their Related Elements[END_REF][START_REF] Cherenok | [END_REF][29][30][31]. Some of them were also functionalized with an anchoring arm to be immobilized on solid supports [7,8,13]. However, none of these phosphoruscontaining calixarenes were used to extract the uranyl ion.

Consequently, the purpose of the present work was also to provide bifunctional ligands which could simultaneously bind uranium and be immobilized on solid supports.

In this work, the synthesis of two phosphorus-containing calix [4]arenes in the 1,3 alternate conformation bearing both phosphonic acid and N-hydroxysuccinimide ester entities is reported.

Their incorporation into a macromolecular matrix is also demonstrated.

Results and discussion

Conception of uranyl cation immobilizing agents: Synthesis of several (phosphonic acid)calix[4]arenes

Phosphorus containing calixarenes modified to provide an N-hydroxysuccinimide moiety able to react with NH 2 groups of a solid support were synthesized. As it is established that the 1,3alternate conformation of calixarenes seems to be the most favorable for metal complexation [32][33][34], the synthesis was designed to produce the new calixarenes with this preferred conformation.

Calixarenes 6 and 9 were obtained via a five-step synthesis. The synthetic pathway is illustrated in Fig. 4.

[insert figure 1 about here]

Calix [4]arene 1 was first O-alkylated in the presence of K 2 CO 3 with 1 equivalent of methoxyethoxy-p-toluenesulfonate to obtain the monoalkoxycalix [4]arene 2 in a 68 % yield. The cone conformation, stabilized by hydrogen bonds, was revealed by the presence of two AB systems at 4.49 and 3.48 ppm (J = 13.0 Hz) and at 4.31 and 3.46 ppm (J = 13.0 Hz), in the 1 H- Calixarenes 5 and 8 were obtained by transesterification using trimethylbromosilane and subsequent hydrolysis of the trimethylsilylesters in a nearly quantitative yield. Finally, the activation of the carboxylic groups was performed using 2 equivalents of NHS and 2 equivalents of EDC to obtain calixarenes 6 and 9 with a 64 % and a 45% yield respectively. For all these compounds, the absence of any AB system in the 1 H NMR spectra confirmed that the 1,3alternate conformation was maintained.

Immobilization of calixarenes 6 and 9 on a gel column

Immobilization was performed by incubating the Ultralink hydrazide gel (15 µmol NH 2 functions per g) with calixarenes quantities corresponding to 20% of the total amount of NH 2 groups.

Since the calixarenes synthesized in this paper are based on a succinimide ester that is commonly used for reactive amine crosslinking, the ester hydrolysis is a limiting factor. Two buffers were tested for the coupling: a HEPES buffer (pH 8.0) and a borate buffer (pH 9.2). A higher pH was shown to favor the nucleophilic attack to the detriment of the ester hydrolysis, and pH 9.2 was thus used in further experiments.

The repeatability of the coupling was then evaluated on 2 different gels using compound 6. An average value of (32 ± 2)% (% mol) of coupled calixarene 6 was found.

The same experiment conducted with calixarene 9 led to 28.5% (% mol), which seems to indicate that the alkyl chain length has no influence on the coupling.

Improvement of the coupling extent was investigated by performing successive calixarene 6 additions and incubations. Results are reported in Table 1 and show that a maximum of 2 µmol calixarene 6 / g gel is obtained.

Immobilization of uranyl ions on a gel column

Assessment of the complexation efficiency on a modified gel was performed. 1 g of the Ultralink hydrazide gel coupled with 2 µmol calixarene 6 / g was incubated overnight in the presence of 5 equivalents of uranyl acetate. A similar experiment was also conducted with the same quantity of a non-coupled gel. The uranyl cation content in the eluates was quantified and found to be 7.5 µmol for the eluates of the gel bound with calixarene 6 and 9.9 µmol for the blank.

Consequently, (2.4± 0.1) µmol uranyl cation / g were immobilized on the modified gel, which corresponds to a 100% immobilization (refering to the calixarene). For the non-coupled gel, the immobilized concentration was found to be as low as (0.1± 0.1) µmol uranyl cation / g. It seems then that the immobilization of the uranyl cation occurs through the formation of a 1:1 (uranyl:calixarene) complex. The total release of the uranyl cation from the blank gel shows the absence of non specific interactions and reinforces this assumption.

Conclusion

The synthesis of new phosphorous-containing calix [4]arenes bearing phosphonic acid functions on one side and NHS activated carboxylic functions on the other side, was achieved. With the aim of providing solid supports, the coupling of these compounds to a solid support was performed. A coupling extent of 2 µmol calixarene 6 / g gel was obtained. Under the used conditions, a quantitative immobilization of uranyl cations was shown to occur through the formation of an 1:1 UO 2 2+ :calixarene 6 complex.

Further studies will be performed to improve the coupling extent of calixarene 6 on such support. However, the use of these heterofunctional macrocycles for many other usages into supramolecular devices could be further envisioned.

Experimental 1. Synthesis

All reagents and solvents were commercial and were used without further purification. Reagents for the synthesis were all Sigma-Aldrich and Prolabo products Calix [4]arene was prepared according to the literature [35]

Synthesis of 1,3-[di-(oxamethyl-phosphonic acid)], 2-(N-succinimide-4-oxabutyrate), 4-(methoxyethoxy)-calix[4]-arene (6).

Mono-methoxyethoxy-calix[4]arene (2).

A suspension of calix [4] Yield: 0.82 g (64 %), mp 109-111 °C.

m/z = 855.1 [MH + ].

Synthesis of 1,3-[di-(oxapropyl-phosphonic acid)], 2-(N-succinimide-4-oxabutyrate), 4-(methoxyethoxy)-calix[4]arene (9).

Diethylphosphonoylpropoxy-p-toluenesulfonate (b).

Diethyl 3bromopropylphosphonate (5.18 g, 20.0 mmol) and silver p-toluenesulfonate (11.16 g, 40.0 mmol) were dissolved in 200 mL acetonitrile and stirred at room temperature for 3 days. The solution was then filtered and the solvent was evaporated to dryness under reduced pressure.

The residue was then taken up in CH 2 Cl 2 (400 mL) and water (400 mL). The organic layer was recovered and dried over anhydrous Na 2 SO 4 . After removal of the solvent, the residue was purified by column (SiO 2 , eluent: CH 2 Cl 2 /acetone 80/20 v/v). Coumpound b was recovered as a viscous liquid.

Yield: 1.75 g ( 25 %).

Coupling on the solid support

A column was packed with 1 g Ultralink hydrazide gel according to the protocol described by the manufacturer and conditionned with a 50 mM sodium borate buffer at pH 9. 40 mg/mL solutions of compound 6 and compound 9 were prepared in DMSO. Five successive additions of these solutions were performed in order to obtain a final concentration of 3 µmol/g calixarene and the coupling was performed during 2.5 h under agitation.

The column was then washed with 2 x 2 mL coupling buffer and 2 mL coupling buffer containing 1M NaCl and the eluates were analyzed using a fluorimeter CARY-Eclipse (Varian) at λ exc = 268 nm and λ em = 310 nm. λ exc and λ em were previously determined from a calixarene solution and the calixarene amount in the eluates was quantified from a calibration curve (0; 1.86; 3.70; and 7.36 x 10 -4 M).

The calixarene content was determined from the mass-balance, by difference between the initial number of moles of calixarene and that found in the eluate fractions.

Uranyl cation binding

  With the aim of developing supports for uranyl cation immobilization, new 1,3alternate bearing both phosphonic acid functions as chelating site and Nsuccinimide-4-oxabutyrate as anchoring arm were synthesized. These compounds were proved to be coupled on a hydrazide gel and to complex uranium successfully.

Formatted 3 uranyl

 3 cation complexation. The solvent extraction of uranium(VI) was widely studied by neutral

  Deleted: the presence of calixarene on the gel allowsDeleted: , which is likely to

  44. Mass spectrum (MALDI-TOF): m/z = 755.2 [M-H] -. Compound 5 (1.14 g, 1.5 mmol), EDC [1-Ethyl-3-(3dimethyl-aminopropyl)-carbodiimide (0.58 g, 3.0 mmol) and NHS (N-hydroxysuccinimide) (0.35g, 3.0 mmol) were dissolved in DMF (90 mL) and the mixture was stirred overnight at room for 3 h at 55°C. After removal of the solvent, the residue was treated with acetonitrile to obtain compound 6 as white solid.

Figure 1 :

 1 Figure 1 : Synthetic pathway for phosphonic acid)calix[4]arenes possessing an anchoring arm. Page 17 of 19

  arene 1 (12.73 g, 30.0 mmol) and K 2 CO 3 (2.16 g, 15.6 mmol) in acetonitrile (500 mL) was stirred for 30 min at room temperature under a nitrogen atmosphere. Methoxyethyl p-toluenesulfonate (6.91 g, 12.0 mmol) was then added and the mixture was stirred and refluxed for 4 days. After removal of the solvent, 400 mL of CH 2 Cl 2 and 400 mL of water were added and the mixture was stirred and acidified with HCl 1M. The organic layer was recovered and dried over anhydrous Na 2 SO 4 . After removal of the solvent, compound 2 was purified by column chromatography (SiO 2 , eluent: CH 2 Cl 2 ) and obtained as a white powder. ArH meta ), 6.89 (t, 1H, J = 7.5 Hz, ArH para ), 6.72-6.65 (m, 3H, ArH para ), 4.49 (d, 2H, J = 13.0 Hz, ArCH 2 Ar), 4.35-4.32 (m, 2H, CH 2 CH 2 OCH 3 ), 4.31 ( d, 2H, J = 13.0 Hz, ArCH 2 Ar), 4.04-4.01 (m, 2H, CH 2 CH 2 OCH 3 ), 3.62 (s, 3H, OCH 3 ), 3.48 ( d, 2H, J = 13.0 Hz, ArCH 2 Ar), 3.46 (d, 2H, J = 13.0 Hz, ArCH 2 Ar). Anal. Calcd. for C 31 H 30 O Br(CH 2 ) 3 C(O)OCH 2 CH 3 (2.34 g, 12.0 mmol) was then added and the resulting solution wasrefluxed for 4 days. After evaporation of the solvent in vacuo, the residue was taken up in CH 2 Cl 2 (300 mL) and in water (300 mL) and the resulting mixture was acidified then separated in order to recover the organic layer which was dried over anhydrous Na 2 SO 4 . After evaporation of the solvent, compound 3 was purified by column chromatography (SiO 2 , eluent: CH 2 Cl 2 /acetone 98/2 v/v) and obtained as a white powder. J = 7.5 Hz, ArH para ), 4.42 (d, 2H, J = 13.0 Hz, ArCH 2 Ar), 4.28 (d, 2H, J = 13.0 Hz, ArCH 2 Ar), 4.23-4.16 (m, 4H, COOCH 2 CH 3 + CH 2 CH 2 OCH 3 ), 4.07 (t, 2H, J = 6.1 Hz, CH 2 CH 2 CH 2 COOEt), 3.95-3.92 (m, 2H, CH 2 CH 2 OCH 3 ), 3.56 (s, 3H, CH 2 OCH 3 ), 3.38 (d, 4H, J = 13.0 Hz, ArCH 2 Ar), 2.90 (t, 2H, J = 7.4 Hz, CH 2 COOEt), 2.41-2.32 (m, 2H, CH 2 CH 2 CH 2 COOEt), Cl 2 (450 mL) at ca. 0°C. The resulting mixture was cooled to room temperature and stirred for 15 h. It was then extracted with 300 mL of acidified aqueous solution and dried over anhydrous Na 2 SO 4 . After removal of the solvent, the residue was purified by column chromatography on silica gel. CH 2 Cl 2 was used as the first mobile phase then CH 2 Cl 2 /acetone residue was taken up in CH 2 Cl 2 (200 mL). 200 mL of water were added and the mixture was acidified with 1 M HCl. The organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The resulting residue was purified by column

	chromatography (SiO 2 , eluent: CH 2 Cl 2 /acetone 90/10 v/v). Compound 4 was obtained as a pure yellow viscous liquid. Yield: 1.69 g (63%). 1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 7.25-7.21 (m, 4H, ArH meta ), 7.11-7.08 (m, 2H, ArH meta ), 6.99-6.96 (m, 2H, ArH meta ), 6.68-6.57 (m, 4H, ArH para ), 4.29-4.17 (m, 6H, 6.65 (t, 2H, 1.29 (t, 3H, J = 7.2 Hz, COOCH 2 CH 3 ). Anal. Calcd. for C 37 H 40 O 7 (%): C, 74.47; H, 6.76. Found ArOCH 2 + COOCH 2 CH 3 ), 4.07 (d, 4H, J = 9.2 Hz, ArOCH 2 P), 3.91-3.88 (m, 2H, CH 2 OCH 3 ),	Deleted: 5 Deleted: 56
		Deleted: 908
	C, 74.68; H, 6.52 3.77-3.64 (m, 8H, POOCH 2 ), 3.59 (s, 3H, CH 2 OCH 3 ), 3.49 (s, 8H, ArCH 2 Ar), 2.53 (t, 2H, J = 7.2
	Hz, CH 2 COOEt), 2.20 (q, 2H, J = 7.2 Hz, CH 2 CH 2 CH 2 COOEt), 1.42 (t, 12H, J = 7.1 Hz,
		Deleted: 119
	1.13. Diethylphosphonoylmethoxy-p-toluenesulfonate (a). A solution of triethylamine (10.12 POOCH 2 CH 3 ), 1.24 (t, 3H, J = 7.1 Hz, COOCH 2 CH 3 ). 31 P NMR (400 MHz, CDCl 3 ) δ (ppm): 21.8.
	Yield: 9.91 g (68%) mp 224-225°C. 1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 9.86 (s, 1H, ArOH), 9.24 7.06 (d, 4H, J = 7.5 Hz, ArH meta ), 6.90 (d, 4H, J = 7.5 Hz, ArH meta ), 6.76-6.71 (m, 2H, ArH para ), w O n l y acetonitrile (100 mL) was stirred for 2 h at room temperature under nitrogen atmosphere. w O n l y (s, 2H, ArOH), 7.12-7.00 (m, 8H, Yield: 3.36 g (56%), mp 165-166 °C. 1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 7.93 (s, 2H, ArOH), F o r P e e r R e v i e g, 100.0 mmol) in CH 2 Cl 2 (50 mL) was added dropwise to a stirred mixture of diethyl(hydroxy-methyl)phosphonate (8.41 g, 50.0 mmol) and p-toluenesulfonyl chloride (10.01 g, 52.5 mmol) in Anal. Calcd. for C 47 H 62 O 13 P 2 (%): C, 62.94; H, 6.97. Found C, 62.76; H, 7.14. F o r P e e r R 1.15. 1,3-[Di-(oxamethyl-phosphonic acid)], 2-(4-oxabutyric acid), 4-(methoxyethoxy)-CH 2 1.14. 1,3-[Di-(oxamethyl-diethylphosphonate)], 2-(ethyl-4-oxabutyrate), 4-(methoxyethoxy)-calix[4]arene (4). A suspension of 1-(ethyl-4-oxabutyrate), 3-(methoxyethoxy)-calix[4]arene 3 (1.79 g, 3.0 mmol) and K 2 CO 3 (4.15 g, 30.0 mmol) in Yield: 0.91 g (98 %), mp 158-159 °C. 1 H-NMR (300MHz, CD 3 OD)  δ (ppm): 7.17 (d, 4H, J = 7.5 e Hz, ArH meta ), 7.05-7.02 (m, 2H, ArH meta ), 6.95-6.92 (m, 2H, ArH meta ), 6.62-6.53 (m, 4H, ArH para ), v i 3.76-3.50 (m, 18H, ArOCH 2 CH 2 + ArCH 2 Ar + ArOCH 2 P + CH 2 OCH 3 ), 3.22 (s, 3H, CH 2 OCH 3 ), e 2.47-2.39 (m, 2H, CH 2 COOH), 2.03-1.92 (m, 2H, CH 2 CH 2 COOH).	Deleted: 55 Deleted: 1 Deleted: 2 Deleted: e Deleted: 826 Deleted: 719 Deleted: 1 Deleted: 407 Deleted: 009 Deleted: 928 Deleted: d Deleted: 0 Deleted: 146
	8

5 (%): C, 77.16; H, 6.27. Found C, 77.43; H, 6.00 1.12. 1-(Ethyl-4-oxabutyrate), 3-(methoxyethoxy)-calix[4]arene (3). Mono-methoxyethoxycalix[4]arene 2 (4.83 g, 10.0 mmol) and K 2 CO 3 (0.72 g, 5.2 mmol) were suspended in acetonitrile (200 mL) and stirred for 1 h at room temperature under a nitrogen atmosphere.

(98/2, v/v). Compound a was recovered as a viscous liquid. Yield: 12.93 g (80%). 1 H-NMR (300MHz, CDCl 3 )  δ (ppm): 7.79 (d, 2H, J = 8.1 Hz, ArH), 7.36 (d, 2H, J = 8.1 Hz, ArH), 4.19-4.09 (m, 6H, SO 3 CH 2 P + POOCH 2 CH 3 ), 2.45 (s, 3H, CH 3 Ar), 1.31 (t, 6H, J = 7.1 Hz, POOCH 2 CH 3 ). Diethylphosphonoylmethoxy-p-toluenesulfonate a (2.03 g, 6.3 mmol) was then added and the mixture was refluxed for 10 days. After filtration of the mixture, the solvent was removed under vacuo and the calix[4]arene (5). Bromotrimethylsilane (5.64 g, 36.83 mmol) was added to a solution of compound 4 (1.10 g, 1.23 mmol) in 20 mL acetonitrile. The reaction mixture was stirred for 24 h at room temperature under nitrogen atmosphere. It was then evaporated under reduced pressure and a mixture of 10 mL of methanol/water (50/50, v/v) was added to the residue. The resulting solution was stirred at room temperature overnight. After removal of methanol and water, the residue was evaporated twice with 10 mL of dry toluene and filtered to obtain compound 5 as a pink solid. 31 P NMR (400 MHz, CD 3 OD) δ (ppm): 19.9. Anal. Calcd. for C 37 H 42 O 13 P 2 (%): C, 58.73; H, 5.59. Found C, 58.47; H, 5.

  1 H-NMR (300MHz, DMSO-d 6 )  δ (ppm): 7.24 (d, 4H, J = 7.4 Hz, ArH meta ), 7.02-6.99 (m, 2H, ArH meta ), 6.93-6.90 (m, 2H, ArH meta ), 6.57-6.52 (m, 4H,

	ArH para ), 3.75-3.55 (m, 12H, ArOCH 2 CH 2 + ArCH 2 Ar), 3.37 (s, 3H, CH 2 OCH 3 ), 3.03-2.89 (m, 6H,
	ArOCH 2 P + CH 2 OCH 3 ), 2.71 (s, 4H, NHS), 1.99-1.95 (m, 2H, CH 2 COONHS), 1.76-1.71 (m, 2H,
	CH 2 CH 2 COONHS).	31 P NMR (400 MHz, DMSO-d 6 ) δ (ppm): 16.4. Anal. Calcd. for C 41 H 45 NO 15 P 2
	(%): C, 57.68; H, 5.31; N, 1.64. Found C, 57.39; H, 5.45; N, 1.81. Mass spectrum (MALDI-TOF):

  Hz, ArH), 4.12-3.99 (m, 6H, CH 3 ArSO 3 CH 2 + POOCH 2 CH 3 ), 2.44 (s, 3H, CH 3 Ar), 2.01-1.87 (m, 2H, SO 3 CH 2 CH 2 CH 2 P), 1.81-1.69 (m, 2H, SO 3 CH 2 CH 2 CH 2 P), 1.29 (t, 6H, J = 7.1 Hz, POOCH 2 CH 3 ). ArH meta ), 7.25-7.22 (m, 1H, ArH meta ), 7.10-7.07 (m, 2H, ArH meta ), 7.01-6.88 (m, 4H, ArH meta ), 6.49-6.42 (m, 2H, ArH para ), 6.32-6.26 (m, 2H, ArH para ), 4.21-4.10 (m, 10H, ArOCH 2 + COOCH 2 CH 3 ), 4.05 (d, 2H, J = 13.0 Hz, ArCH 2 Ar), 3.92-3.57 (m, 12H, POOCH 2 CH 3 + ArCH 2 Ar), 3.45 (s, 3H, CH 2 CH 2 OCH 3 ), 3.09 (d, 2H, J = 13.0 Hz, ArCH 2 Ar), 2.61-2.43 (m, 2H, CH 2 CH 2 OCH 3 ), 2.23-2.14 (m, 4H, CH 2 POOEt + CH 2 COOEt), 1.96-1.84 (m, 2H, CH 2 POOEt), 1.76-1.53 (m, 6H, CH 2 CH 2 CH 2 POOEt + CH 2 CH 2 CH 2 COOEt), 1.43-1.34 (m, 12H, POOCH 2 CH 3 ), 1.26 (t, 3H, J = 7.1 Hz, COOCH 2 CH 3 ). 31 P NMR (400 MHz, CDCl 3 ) δ (ppm): 34.1 and 33.1 ppm. ArOCH 2 + ArCH 2 Ar + CH 2 CH 2 OCH 3 ), 3.31 (s, 3H, CH 2 OCH 3 ), 2.12-1.98 (m, 2H, CH 2 COOH), 1.72-1.45 (m, 10H, CH 2 CH 2 CH 2 POOH + CH 2 CH 2 CH 2 COOH). The synthesis was performed as for compound 6. COONHS), 1.57-1.21 (m, 10H, CH 2 CH 2 CH 2 POOH + CH 2 CH 2 CH 2 COONHS).

	mL. Column chromatography (SiO 2 , eluent: CH 2 Cl 2 /acetone 80/20 v/v). Compounds 7 and 7'
	were obtained as yellow viscous liquids. 1.24. 1,3-[Di-(oxapropyl-phosphonic	acid)],	2-(N-succinimide-4-oxabutyrate),	4-
					Deleted: 526
	Compound 7 (1,3-alternate), yield: 0.53 g (23 %). (m, 2H, ArH meta ), 7.02-7.00 (m, 6H, ArH meta ), 6.75-6.70 (m, 4H, ArH para ), 4.21-4.09 (m, 10H, 1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 7.09-7.06 (methoxyethoxy)-calix[4]arene (9). Calix[4]arene derivative 8: 0.22 g, 0.27 mmol; EDC: 0.10 g, 0.54 mmol; NHS: 0.06 g, 0.54
	ArOCH 2 + COOCH 2 CH 3 ), 3.75-3.55 (m, 16H, POOCH 2 CH 3 + ArCH 2 Ar), 3.37-3.35 (m, 2H, mmol; DMF: 16 mL. Brown solid, yield: 0.11 g (45%) ; mp > 290 °C. 1 H-NMR (300MHz, DMSO-
	CH 2 CH 2 OCH 3 ), 3.34 (s, 3H, CH 2 CH 2 OCH 3 ), 2.26 (t, 2H, J = 7.5 Hz, CH 2 CH 2 CH 2 COOEt), 1.92-d 6 ) δ (ppm): 7.13-6.94 (m, 8H, ArH meta ), 6.79-6.69 (m, 4H, ArH para ), 3.66-3.46 (m, 18H, ArOCH 2
	1.66 (m, 10H, CH 2 CH 2 CH 2 POOEt + CH 2 CH 2 CH 2 COOEt), 1.37 (t, 12H, J = 7.1 Hz, + ArCH 2 Ar + ArOCH 2 CH 2 OCH 3 ), 3.36 (s, 3H, CH 2 OCH 3 ), 2.71 (s, 4H, NHS), 1.99-1.88 (m, 2H,
	POOCH 2 CH 3 ), 1.30 (t, 3H, J = 7.1 Hz, COOCH 2 CH 3 ). CH 2 31 P NMR (400 31 P NMR (400 MHz, CDCl 3 ) δ (ppm): 33.5
	F o r ppm. Anal. Calcd. for C 51 H 70 O 13 P 2 (%): C, 64.27; H, 7.40. Found: C, 64.17; H, 7.29. Mass spectrum (MALDI-TOF): m/z = 975.4 [MNa + ]. Compound 7' (partial cone), yield: 0.43 g (19 %). 1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 7.35-7.33 MHz, DMSO-d 6 ) δ (ppm): 32.1. Anal. Calcd. for C 45 H 53 NO 15 P 2 (%): C, 59.40; H, 5.87; N, 1.54. F Found C, 59.18; H, 5.60; N, 1.60. Mass spectrum not available due to the poor solubility in o r solvents compatible with mass spectrometry.	Deleted: 4
	P e e r R e P e e r R (m, 1H, Anal. Calcd. for C 51 H 70 O 13 P 2 (%): C, 64.27; H, 7.40. Found C, 64.17; H, 7.29 e		Deleted: 2 Deleted: 3
	v i e 1.23. 1,3-[Di-(oxapropyl-phosphonic acid)], 2-(4-oxabutyric acid), 4-(methoxyethoxy)-calix[4]arene (8). The synthesis was performed as for compound 5. v i e	Deleted: 2 Deleted: d
	w Calix[4]arene derivative 7: 0.29 g, 0.3 mmol; bromotrimethylsilane: 1.38 g, 9.0 mmol; acetonitrile: 10 mL; methanol/water (50/50, v/v): 5 mL. Pink solid, yield: 0.24 g (98%); mp > 290 w	Formatted: Italian Italy Deleted: 286 Deleted: 378
	1.22. (methoxyethoxy)-calix[4]arene (7 and 7'). The synthesis was performed as for compound 4. 1,3-[Di-(oxapropyl-diethylphosphonate)], 2-(ethyl-4-oxabutyrate), O n l 4-°C. 1 H-NMR (300MHz, CD 3 OD) δ (ppm): 7.15-7.03 (m, 8H, ArH meta ), 6.92-6.77 (m, 4H, ArH para ), 3.84-3.49 (m, 18H, 31 P NMR (400 O n l	Deleted: d Deleted: 2 Deleted: 239
	Calix[4]arene derivative 3: 1.43g, 2.4 mmol; K 2 CO 3 : 3.32g, 24.0 mmol; acetonitrile: 100 mL; diethylphosphonoylpropoxy-p-toluenesulfonate b: 1.77 g, 5.0 mmol; CH 2 Cl 2 : 200 mL; H 2 O: 200 60.46; H, 6.13. y MHz, CD 3 OD) δ (ppm): 33.5 ppm. Anal. Calcd. for C 41 H 50 O 13 P 2 (%): C, 60.59; H, 6.20. Found C, y	Deleted: 17 Deleted: 766

1 H-NMR (300MHz, CDCl 3 ) δ (ppm): 7.78 (d, 2H, J = 8.2 Hz, ArH), 7.34 (d, 2H, J = 8.2

Table 1 :

 1 Successive couplings of calixarene 6 on the same gel

	Page 19 of 19
	Experiment number	Addition of calixarene 6	Calixarene 6 found in the	% calixarene 6
		(µmol)	eluate (µmol)	coupled to the gel
	1	2.99	1.99	33.5
	2	2.99	1.97	34.1
	3	6.00	6.15	-

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

Acknowledgment

The authors would like to thank the TOXNUC-E French program for financial support.

sodium acetate buffer pH 4.0. 5 x 20 µL of a 0.1 M uranyl acetate solution ( i.e 10 µM) were then added to the column and contacted overnight under agitation at room temperature. The supernatant was recovered and the column was then rinsed using 3 x 2 mL 50 mM acetate buffer pH 4 and 3 x 2 mL 50 mM HEPES buffer pH 7.4

UV spectra of uranyl solutions, recorded on a CARY 50 (Varian), showed a maximum peak between 222 and 234 nm, correlated to the uranyl concentrations in the solutions. A calibration curve ( 0; 1.5; 3; and 6 x 10 -4 M) was used to quantify the uranyl amount in both supernatant and eluates. The amount of gel sorbed uranyl cation was determined by difference between its initial quantity and that measured in the liquid fractions.