Submerging a Biomimetic Metallo‐Receptor in Water for Molecular Recognition

A Journal of

Accepted Article

Title: Submerging a Biomimetic Metallo-Receptor in Water for Molecular Recognition: Micellar Incorporation or Water Solubilization? A Case Study

Authors: Solène Collin, arnaud parrot, Lionel Marcelis, emilio Brunetti, ivan jabin, gilles Bruylants, Kristin Bartik, and Olivia Reinaud

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To be cited as: Chem. Eur. J. 10.1002/chem.201804768

Link to VoR: http://dx.doi.org/10.1002/chem.201804768

Supported by Chemistry - A European Journal 10.1002/chem.201804768

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Submerging a Biomimetic Metallo-Receptor in Water for Molecular Recognition: Micellar Incorporation or Water Solubilization? A Case Study Solène Collin,[a] Arnaud Parrot,[a] Lionel Marcelis,[b] Emilio Brunetti,[b,c] Ivan Jabin,[c] Gilles Bruylants,[b] Kristin Bartik,*[b] and Olivia Reinaud*[a]

Abstract: Molecular recognition in water is an important topic but solvents, stem from the intrinsic properties of this solvent.[2] challenging task due to the very competitive nature of the medium. Indeed, it provides a very competitive medium for electrostatic The focus of this study is the comparison of two different strategies interactions, and particularly for the directional H-bonding for the water solubilization of a biomimetic metallo-receptor based on interactions. It is thus necessary to associate other types of the poly(imidazole) resorcinarene core. The first one relies on a new stabilizing effects in order to extract analytes from water. The synthetic path for the introduction of hydrophilic substituents on the hydrophobic effect[3] can be exploited for analytes that present an receptor, at a remote distance from the coordination site. The second apolar component in their structure.[4,5] For highly polar one consists in the incorporation of the organosoluble metallo- compounds, such as small anions,[6,7] coordination to a metal receptor into dodecylphosphocholine (DPC) micelles that mimics the cation[8–10] can provide an efficient way to shift the equilibria proteic surrounding of the active site of metallo-enzymes. The towards the formation of a host-guest adduct, i.e. a metal resorcinarene ligand can be transferred into water via both strategies complex.[11] However, water is also a strong competitor in the case where it binds ZnII over a large pH window. Quite surprisingly, very of metal-coordination due to its good donor ability in its neutral similar metal ion affinity, pH response and recognition properties were form or as hydroxide. In Nature, and in particular in the case of observed with both strategies. The systems, behave as remarkable proteins, this problem is circumvented[12] as the metallo active receptors for small organic anions in water, near physiological pH. sites are generally associated to a well-defined hydrophobic These results show that, provided the biomimetic site is well cavity that also plays a major role in substrate recognition. structured and presents a recognition pocket, the micellar Inspired by this design, we are developing biomimetic cavity- environment has very little impact on both metal ion binding and guest based metal complexes as a tool for molecular recognition.[13] hosting. Hence, micellar incorporation represents an easy alternative These so-called “funnel” or “bowl” complexes have in common the to difficult synthetic work, even for the binding of charged species presence of a hydrophobic cavity in the vicinity of a coordinated

(metal cations or anions), which opens new perspectives for metal ion that act in synergy for guest binding. The funnel Manuscript molecular recognition in water, either for sensing, transport or complexes, based on the flexible calix[6]arene core, have been catalysis. shown to display remarkable recognition properties towards neutral guests presenting both, good donor ability for metal ion binding and good shape complementarity to the cone cavity (Figure 1, left).[14–17] Surprisingly, most of these complexes were Introduction observed to be reluctant to anion binding, which made them unusually selective.[18] This was attributed to electrostatic The study of molecular recognition processes in water is a field of repulsion by the oxygen-rich narrow rim as a second coordination great interest, not only for the elaboration of efficient and selective sphere. In contrast, the rigid bowl-shaped resorcinarene-based sensors for environmental analyses or the development of green metal complexes, offering very different second coordination [1] catalysts, but also for the elucidation of biological processes. sphere and shape, display strong affinity to small anionic The major difficulties encountered in water, compared to organic guests.[19] Our most recently described bowl complex, based on the new [20] ligand Rim4, is depicted in Scheme 1. In this system, the [a] S. Collin, A. Parrot, and O. Reinaud resorcinarene is functionalized at its large rim with four imidazole Laboratory of Pharmacological and Toxicological Chemistry and II Biochemistry arms of which three firmly hold the Zn ion at the entrance of the Université Paris Descartes bowl-shape cavity, whereas the fourth is hemilabile. We showed

45, rue des Saints-Pères / 75006 Paris / FRANCE that this hemilabile arm assists the deprotonation of a neutral Accepted E-mail: [email protected] guest molecule, which can then bind as an anion to the metal [b] L. Marcelis, E. Brunetti, G. Bruylants, and K. Bartik Engineering of Molecular Nanosystems center. It was also shown to promote the selective base-catalyzed Université Libre de Bruxelles hydration of acetonitrile into acetamide, thus coined as “a third Avenue F. D. Roosevelt 50, CP165/64, B-1050 Brussels / BELGIUM degree of biomimetism”.[20] In our quest towards more E-mail: [email protected] biomimetism, the following aspects were then considered: i) the [b] E. Brunetti, and I. Jabin. Laboratory of Organic Chemistry solvent for the natural systems, i.e. enzymes, is water; and ii) in Université Libre de Bruxelles soluble enzymes, the active site is surrounded by a large proteic Avenue F. D. Roosevelt 50, CP160/06, B-1050 Brussels / BELGIUM structure, mostly hydrophobic in its heart, whereas a polar surface Supporting information for this article is given via a link at the end of insures its water-solubility. the document.

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II (-) Scheme 1. Schematized structure and general host-guest behavior of complex Rim4Zn in MeCN. G stands for an anionic guest ligand and S stands for a solvent or water molecule.

To face this new aspect of biomimetism, two different strategies amines in water.[21,22] The second strategy relies on the II have been considered for the water-solubilization of the Rim4Zn incorporation of the organo-soluble metal complex into micelles. system. The first strategy consists in the implementation of This has been exemplified previously for a metallo-receptor with hydrophilic moieties to the macrocyclic structure. This requires an uranyl-salophen complex for anion binding.[23,24] More recently, careful synthetic design to avoid interaction between the we have reported the successful incorporation of a calix[6]arene hydrophilic moieties and the metal ion and often leads to laborious ZnII funnel complex in DPC micelles and have shown that the synthetic work. Such a strategy has been previously successfully system keeps its hosting properties towards amines at applied to various funnel complexes as illustrated by physiological pH. However, none of these funnel complexes tris(imidazole)ZnII-based and trenCuII-based calix[6]arene recognize anions, even lipophilic ones.[25] systems that behave as selective receptors for lipophilic primary

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Figure 1. Left: A water-soluble funnel complex. Right: two strategies for water solubilizing a resorcinarene-based ZnII receptor, subject of this article.

Here, we report a study that compares the two different strategies Synthesis of the water-soluble ligand WRim4 for the water-solubilization of a resorcinarene-based metallo- We have recently described a strategy to transform an organo- receptor (Figure 1, right) for the recognition of small organic soluble ligand based on a resorcinarene macrocycle presenting [26] guests that bind in their anionic form t (Figure 1, right): direct three imidazole groups (so-called Rim3) into a water-soluble water-solubilization of the ligand into water, which will then one thanks to the introduction of quaternary ammonium groups Accepted [27] surrounds the metallo receptor, or encapsulation into a zwiterionic as “feet”. The synthesis of WRim4 followed a similar strategy micelle that will mimic the hydrophobic environment provided by (Scheme 2), but using pyridinium as water-solubilizing feet, the proteic backbone of natural systems. according to a procedure adapted from one previously reported by Rebek et al. for deep cavitands.[28] The tetrabromoderivative 1 was obtained according to a Results and Discussion previously reported route, within three steps starting from resorcinol.[29] After protection of the OH groups as silyl ethers, the

FULL PAPER resulting compound 2 was treated with n-butyllithium and paraformaldehyde to provide the corresponding tetra(alcohol) derivative 3. The four imidazole groups were grafted at the large rim using 2-chloromethyl-1-methyl-1H-imidazole with NaH to give compound 4. Deprotection of the OH groups afforded product 5. This later was reacted with chloromethylsulfonate (MsCl) and then heated in pyridine, giving target compound WRim4 as its 1 chloride salt. Its H NMR spectra in CD3OD and D2O are displayed Figure 2 and compared to that of Rim4 in CDCl3.

Scheme 2. Synthesis of the water-soluble ligand WRim4. (i) TIPSCl, imidazole, DMF, 82%; (ii) nBuLi, paraformaldehyde, THF, -78 °C then rt, 45%; (iii) N- methylchloromethylimidazolium hydrochloride, NaH, DMF, 0 °C then rt; (iv) TFA, THF/H2O (1:1), 93% overall yield from 3; (v) MsCl, Pyridine, rt then 100 °C, 60%.

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1 Figure 2. H NMR spectra of Rim4 systems in different media. From top to bottom: A) WRim4 in CD3OD (500 MHz); B) WRim4 in water (4 mM, pD = 7.4, 500 MHz); C) MRim4 in water (0.5 mM, pD = 7.4, 20 mM DPC, 600 MHz); D) Rim4 in CDCl3 (600 MHz).

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1 As with the Rim4 parent system in CDCl3, the H NMR spectra of undergoes progressive deprotonation up to pD 7. Above this pD WRim4 present sharp and well-defined peaks in CD3OD as well the NMR spectra did not change, thus attesting to the as in D2O that attests to a C4v-symmetrical structure (Figure 2). A deprotonation of all four imidazole donors. The displacement of comparison of the spectra in different solvents gives some the chemical shifts according to pD led to an average pKa of 5.7 insights into the relative environment of the protons. Several for the four imidazole arms. features can be evidenced: all imidazole peaks display very Incorporation of Rim4 into micelles similar values (between 6.8 and 7.1 ppm, peaks labeled a and b). We recently described a very efficient way to incorporate a In contrast, the aromatic protons belonging to the resorcinarene macrocyclic cationic metallo-receptor into micelles.[25] The structure (labeled h) appears at very different δ shifts, receptor, a calix[6]arene-based ZnII complex,[30] was successfully emphasizing the different environments offered by the solvents. incorporated into dodecylphosphocholine (DPC) micelles and the Varying the pD from 2 up to 11 evidenced a shift of the incorporated complex was observed to be stable over a wide pH resonances corresponding to the imidazole arms due to different range. We tested the same strategy with Rim4. Due to its protonation states (Figure 3 and S5). As for the previously insolubility in water as a neutral compound, it was incorporated reported WRim3 ligand, WRim4 is fully protonated below 3.8, and into the micelles at low pH, where it is under its protonated form.

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1 Figure 3. Evolution of the H NMR spectra of MRim4 (0.5 mM, 20 mM DPC, left) and WRim4 (2.5 mM, right) in D2O as a function of pD (the full spectra are displayed in Figures S5-6). From acidic to basic conditions: tetraprotonated ligand (blue dots), the fully deprotonated ligand (green dots) and mixtures of protonated products between the pH extremes. The highlighted peaks correspond to, from left to right: Hb and Ha (imidazoles) and Hf (methylene bridges). The peak that does not shift with pH corresponds to the aromatic proton of the resorcinol units (Hh, see assignments in Figure 2). Right: normalized variation with pH of the chemical shifts of the dotted signals.

The corresponding NMR spectrum attested to its solubilization in remains fully protonated up to pD of approximately 4, and a fully protonated state. Once incorporated, the ligand remained undergoes complete deprotonation at pD = 8 (Figure 3 and S6). inside the micelles at neutral and high pH where it is in its basic The average pKa of the imidazole arms for MRim4 is 6.3, which 1 form (Figure 2c). A H NMR study at different pH revealed that it represents a shift of 0.6 pH units compared to WRim4. Accepted

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II II Scheme 3. Two pathways to obtain MRim4Zn . a) Incorporation of the ligand at low pH into DPC micelles followed by neutralization of the medium and Zn - complexation; b) Direct incorporation of the isolated organo-soluble complex at pH 7. S is a molecule of organic solvent. W stands for H2O or HO

II Zn complexation to WRim4 and MRim4 The ZnII complexes were readily obtained upon addition of one II equiv. of Zn to millimolar solutions containing either WRim4 or II MRim4 in water at neutral pH. The Rim4Zn complex could also be directly and efficiently incorporated into DPC micelles at neutral pH (Scheme 3), and it gives rise to an NMR signature II identical to the one of MRim4 at neutral pH after addition of Zn . II DOSY spectra recorded for the MRim4Zn system clearly show Manuscript the same diffusion coefficient for the surfactant and receptor (Figures S10), proving the incorporation of the ligand. The stability II of the two Rim4Zn systems was monitored as a function of pD by 1H NMR spectroscopy and spectra are presented in Figure 4. At low pDs (<4), the NMR signature corresponds to the protonated ligand, which indicates that zinc is not coordinated. As the pD is raised, the resonances of the imidazole arms start to shift up-field, which is indicative of the formation of the ZnII complex. At intermediate pHs (6–8), the spectra indicate full complexation of ZnII. At high pH (>8) decomplexation starts to occur as indicated by the emergence of new peaks in the aromatic region, which corresponds to the free ligand, most probably following to formation of zinc hydroxide.[31] II, The complexes are stable over a wide pH range. For WRim4Zn (2.5 mM) the 50% stability window is [4.9–8.7], whereas for II MRim4Zn (0.5 mM complex; 20 mM DPC) it is [5.6–9.8] (see Figures S8-11). The pD window of stability observed for the

micellar system sits at slightly more basic values, which is Accepted 1 consistent with the slightly higher pKa value of the free ligand Figure 4. Evolution of the H NMR spectra of MRim4 (0.5 mM, left) and WRim4 II when incorporated in the micelles (see Table 1). Due to the (3.5 mM, right) in D2O as a function of pD in the presence of 1 equivalent of Zn multiple equilibria, the NMR studies did not allow to identify the (the full spectra are displayed in Figures S8 and S11). From basic to acidic conditions: the neutral ligand (green dots), the zinc complex (red dots), the fully protonation state of the guest (water or hydroxide), nor the protonated ligand (blue dots). The highlighted peaks correspond to, from left to possible protonation of one imidazole arm of the Zn complexes, right: Hb and Ha (imidazoles) and Hf (methylene bridges). The unmarked peak, as previously evidenced in MeCN (vide supra). which does not shift, corresponds to the aromatic proton of the resorcinol units (Hh, see assignments in Figure 2). Note: in the 7.6–9.4 pH window, the aromatic proton of MRim4 overlaps with an imidazole peak.

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The binding constants for ZnII at pH 7 were measured by ITC in the bowl cavity. The presence of two signals in the high field (Figure S9, S12) and the corresponding thermodynamic region, associated to two sets of three signals in the aromatic parameters are reported in Table 1, together with those previously region (one for the macrocycle and two for the imidazoles), is [20] reported for Rim4 in acetonitrile for comparison purposes. likely due to the co-existence of a solvated form of the host-guest 2+ In water, the binding constants of Zn to WRim4 and MRim4 are system where a water ligand replaces one imidazole arm (see the 4 -1 II surprisingly similar (K’Zn ∼ 10 M ) and with only little differences structures displayed in Figure 5), as evidenced with Rim4Zn in in the enthalpic and entropic components, in spite of the very MeCN solutions containing water. The corresponding K’OAc values, different micro-environments surrounding the resorcinarene- estimated by integration of the NMR signals for both systems, are 4 -1 based ligands. The K’Zn values measured in water are only one very similar (1–2 x 10 M , see Figure 5 and Table 2). This order of magnitude lower than the one measured in MeCN with suggests that there is little influence of the environment for the Rim4 despite the stronger solvation of the metal ion. The stronger endo coordination of acetate, and this can be attributed to the competition with the water solvent as a ligand compared to MeCN presence of the bowl structure that surrounds the guest. Varying is well reflected in the enthalpic contribution, ∆H°’Zn, which is the pD evidenced an optimal value of 7.4 for acetate binding for much more favorable in MeCN than in water (–70 vs. ca. –10 both systems (Figure 5). At high pDs, the stability window is -1 kJmol ). The entropic component, ∆S°’Zn, is positive for the increased by ca. 1 pD unit compared to the metallo-receptor in systems in water, which stands in contrast with what is observed the absence of guest, with a significant advantage for the micellar in MeCN where it is negative (–100 JK-1mol-1). The loss of entropy system (10.5 vs. 9.8 for 50% of complexation, see Tables 1 and observed for the system in MeCN is a consequence of the higher 2). order state of the ligand when the metal complex is formed since The effect of the carboxylate length on coordination was also the imidazole arms are no longer able to freely move around. The investigated. Addition of formate triggered a change in the NMR II positive ∆S°’Zn values observed in water suggest that desolvation signature of MRim4Zn that attested to its coordination, most likely of zinc and related water molecules release are entropically in the endo position in view of its size and its behavior with II 2 -1 decisive. Moreover, in the case of WRim4, the hydrophobic effect Rim4Zn in MeCN (K’HCO2- = 5 x 10 M ). No coordination was minimizes ligand exposure to water when folding and wrapping however evidenced with WRim4 under similar experimental around the ZnII ion. In micelles, a local solvophobic effect due to conditions, which is attributable to formate’s high solvation and the interpenetration of the lipophilic chain of the surfactant and the small size, not optimal for cavity filling. With the larger propionate opened, unfolded bowl-cavity of free Rim4 might be responsible anion, no modification of the NMR spectra was observed for both -1 -1 for the relatively high value of ∆S°’Zn (+60 JK mol ). This study systems with up to 50 equiv., which highlights the selectivity of the clearly evidences that, whereas in organic solvents ZnII systems. II II coordination is enthalpically-driven, in water, the entropic Acetylacetonate. The titration of the WRim4Zn and MRim4Zn contribution to ∆G° is dominant in both cases, with surprisingly complexes by acetylacetone at pH 7.4, was monitored by 1H NMR little impact of the presence of micelles. spectroscopy, and the quantitative formation of the corresponding Manuscript Table 1. Binding constants and associated enthalpic and entropic parameters host-guest adduct was observed, with a binding constant higher II 4 -1 for Zn complexation to Rim4 in MeCN, and WRim4 and MRim4 in water at pH than 10 M . The complexes were characterized by a shift of the 7. The values were determined by ITC titration of the ligands (0.25 mM and 0.5 peaks of the imidazole arms and the emergence of a new II mM) by a solution of Zn buffered by HEPES 100 mM and 50 mM for WRim4 II II resonance at –2.35 and –2.20 ppm for WRim4Zn and MRim4Zn and MRim4, respectively (see the ESI and ref [20] for Rim4). respectively, which attests to the inclusion of a methyl group in

Parameters Rim4 MRim4 WRim4 the resorcinarene cavity. The NMR signatures and complexation induced shift (CIS) values are very similar to those previously -1 5 4 4 II K’Zn (M ) (6 ± 2) x 10 (3 ± 1) x 10 (2 ± 1) x 10 reported with Rim4Zn in MeCN in the presence of acetylacetone [20] -1 and triethylamine (Table 2). This indicates that, in water at pH ∆H°’Zn (kJmol ) -70 ± 7 -8.5 ± 2 -11 ± 3 7 as in MeCN, acetylacetone is strongly bound to the metal ion -1 -1 ∆S°’Zn (JK mol ) -100 ± 23 60 ± 10 50 ± 15 thanks to its deprotonation, which leads to the bidentate anionic ligand, acetylacetonate, and the associated displacement of one [a] pD stability (50%) - 5.6 – 9.8 4.9 – 8.6 imidazole arm. In this complex, one guest methyl group sits in the

[a] In this case, the solvent was CH3CN. endo position and the other in an exo position, exposed to the solvent (see the structures displayed in Figure 6). The binding site Host-guest studies of the receptor is thus expansible, as already observed with II [19] Carboxylates. We previously showed that, in MeCN, the Rim4Zn Rim3. The presence of only two resonances for the complex can bind with high affinity one equiv. of acetate in the coordinating imidazoles in the aromatic region indicates that the Accepted endo position and we carried out a comparative study with WRim4 imidazole arms are flipping quickly (relative to the NMR chemical and MRim4 to see if the micro-environment around the metal shift time scale) from a coordinated state to an uncoordinated one, II complex has an impact on the hosting properties of the Zn as previously observed with Rim4 in MeCN. II II complex. The titration of MRim4Zn and WRim4Zn with sodium Varying the pH evidenced efficient recognition over large pH 1 acetate at pD 7.4 was monitored by H NMR spectroscopy (Figure windows for both systems. With WRim4 (0.5 mM), the 50% 5). The spectra evidenced clearly the emergence of new signals stability window for the acetylacetato ZnII complex is [6–10], δ around =–2.4 ppm, attesting to the inclusion of the acetate ion whereas for MRim4 (0.5 mM receptor, 20 mM DPC) it is even

FULL PAPER larger (i.e. 6–11). The fact that 50% of acetylacetonate inclusion Again, very similar thermodynamic parameters were found, ‡ is detected at pH 5.8 indicates a pseudo-pKa = 5.8, which indicating, that in both cases the strong complexation is -1 corresponds to a shift of more than 3 units relative to free essentially enthalpy driven (∆H°’acac=–23 ± 3 kJmol and –24 ± 1 [32] -1 -1 -1 -1 -1 II acetylacetone (pKa = 9). ITC measurements (Figure 6) gave kJmol , ∆S°’acac=8 ± 1 JK mol and 7 JK mol , for MRim4Zn 4 -1 II corresponding K’acac values of 2.8 (± 0.7) x 10 M and 2.4 (± 0.7) and WRim4Zn , respectively). 4 -1 II II x 10 M at pH 7 for MRim4Zn and WRim4Zn , respectively.

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II II 1 Figure 5. Acetate binding to WRim4Zn and MRim4Zn in water (W stands for a water or hydroxide ligand). Left and right: portions of the H NMR spectra recorded II II in D2O at pD 7.4 during the titrations. Conditions: WRim4Zn 0.7 mM in 100 mM HEPES buffer (300 K, 500 MHz); MRim4Zn 0.5 mM, DPC 20 mM in HEPES 50 mM (298 K, 600 MHz). Center: fit for 1:1 binding constants at pD 7.4 and pD stability window of the acetate complexes as measured by integration of the peaks of the endo-bound acetate, at ca. –2.3 ppm (see Figures S17-19 for the corresponding complete NMR spectra).

II 1 Table 2. Data for host-guest properties of different Rim4-based Zn complexes. For MRim4 and WRim4, the binding constants were determined by H NMR spectroscopy at pD = 7.4 for acetate and at pD = 8.4 for acetamide, and at pH 7 by ITC for acetylacetone (see ESI). The pD windows (given for 50% existence) 1 were determined by H NMR. For Rim4 see ref. [20].

Acetate Acetylacetonate Acetamidate

Guest [a] [b] [b] CIS pD window -1 CIS pD window -1 CIS pD window -1 K’pH7.4 (M ) K’pH7 (M ) K’pH8.4 (M ) (ppm) (50%) (ppm) (50%) (ppm) (50%)

4 4 2 MRim4 -4.2 5.5 – 10.5 2.2(±0.5) x 10 -4,5 5.7 – 11.2 2.8(±0.7) x 10 -4,3 7.9 – nd 7(±1) x 10

3 4 2 WRim4 -4.3 5.7 – 9.8 8.2(±1.0) x 10 -4,6 5.8 – 10.3 2.4(±0.7) x 10 -4,4 7.5 – 10.2 4(±1) x 10 Accepted

[c] 4 [c] 4 [c] 3 Rim4 -4.4 - >10 -4,1 - >10 -4,2 - 1.2(±0.5) x 10

[a] The reference δ shift for the free guest corresponds to acetate in water and acetic acid in MeCN. The CIS value corresponds to an average value of the two high field peaks. [b] The reference δ shift for the free guest corresponds to its neutral state. [c] In this case, the solvent was CH3CN.

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II II Figure 6. Complexation of acetylacetonate in water. The binding constant in micelles was obtained by ITC titration of WRim4Zn (Left, 0.8 mM) and MRim4Zn (right, 0.5 mM, DPC 20 mM) by acetylacetone at pH 7 (HEPES 50 mM).

Acetamide. Having evidenced that the systems can bind acetylacetonate at neutral pH in spite of its high pKa value (9), Conclusions acetamide coordination was studied, knowing that it is a poor ligand when neutral (especially in the competitive aqueous The water-soluble version of Rim4, namely WRim4, was medium), but a strong donor when deprotonated. Coordination of successfully synthesized using the “feet” functionalization II II acetamide by WRim4Zn and MRim4Zn was explored as a

strategy. Indeed, the tetrapyridinium salt, readily obtained from Manuscript 1 function of pD by H NMR. The pD of a solution containing WRim4 the hydroxylated precursor, was synthesized within 8 steps from (1 mM), ZnII (2 mM) and acetamide (50 mM) was varied and the resorcinol, with an overall yield of 8% compared to 5 steps and [20] NMR spectra showed the endo complexation of acetamide (δ = – 15% yield for its organosoluble analog, Rim4. The water- 2.35 ppm for its methyl group) in the [7.5 – 10.2] pD window, with solubilization of Rim4 through its incorporation into DPC micelles an optimal pD of 9 for its inclusion. Titration of WRim4 and MRim4 was also successfully achieved. Both strategies lead to mM with acetamide at pD 8.4 allowed to estimate the corresponding concentrations of the ligand in water. Interestingly, the pKa -1 K’MeCONH values as 400 and 700 M , respectively (Figures S22- difference of 0.6 unit for the protonation of the ligand indicates that 24). This is a remarkable result considering the low hydrophobic the micellar environment stabilizes the (partially) protonated form [33] driving force for such a small guest and its high pKa value (16). of Rim4 more than water. This is probably indicative of stabilizing The presence of ca. 50% of endo complex at pD 7.5 indicates a interaction of the cationic form by the phosphate moieties of the pseudo pKa shift of 8 units, which is impressive. Similar pKa shifts DPC micelles. This also accounts for the higher basic pH window (ca. 8 units) were reported for acetamide residues that are (9.8 vs. 8.6) for the coordination of the ZnII cation. Importantly, intramolecularly coordinated to the metal center thanks to a strong both ligands WRim4 and MRim4 display good affinity for zinc that chelate effect with the formation of a 5-membered ring.[34,35] In they readily bind to give rise to a folded structure prone to guest contrast, intermolecular coordination of amides (such as binding. In contrast to the enthalpy driven coordination of ZnII by acetamide or benzylamide) was not observed with these systems the organosoluble system Rim4 in MeCN, it is entropy driven in deprived of cavity. This shows that the effect of the resorcinarene water, where both systems WRim4 and MRim4 allow to stabilize cavity for stabilizing a guest has an impact that is as efficient as a the bowl complexes over a large pH window. The corresponding Accepted strong chelate effect. Looking at the literature, we have not been ZnII complexes bind small organic anions in water, near able to find a comparable affinity for acetamide in water by any physiological pH, with efficiency and selectivity in spite of the other molecular receptors. guest hydrophilicity. Remarkably, they recognize acetamide at physiological pH as an anion in spite of its low acidity (pKa 16). The corresponding pseudo-pKa value of 7.5 gives rise to an impressive pKa shift of 8.5, which is well explained by the presence of a coordination bond with the Lewis acidic ZnII center

FULL PAPER concomitant with the burial of the guest in the resorcinarene cavity. anionic form for acetate, or neutral form for acetylacetone and When compared to simple ZnII complexes, the thermodynamic acetamide) shows also similar K values. Also surprising is the impact of the cavity for the stabilization of a metal-coordined similarity of the pH windows both, for metal and guest binding. deprotonated amido moiety is as efficient as a 5-membered ring The only difference that we essentially observed is a slightly chelate effect. As far as we know, this is the first example of a enlarged pH window (by 0.5–1 pH unit) at basic pHs with micelles, receptor able to bind acetamide as an anion in water, at which is reflected by the difference of pKas of the ligands WRim4 physiological pH. As for the organosoluble version, the selectivity and MRim4. II of guest hosting is cavity-driven by steric effects with two Also striking is the fact that the properties of MRim4 (Zn coordination sites available thanks to the hemilability of one coordination and guest binding) resemble more those of WRim4 imidazole. These hosting properties stand in strong contrast to than those of Rim4 in MeCN. This can be attributed to the major those displayed by calix[6]arene-based funnel complexes, which role played by the solvation of the free ZnII and free guest as, once are excellent receptors for neutral guests such as amines, in spite bound to the resorcinarene tris-imidazole structure, the metal and of their protonated state at physiological pH, but are reluctant to guest sit in the environment provided by the bowl-shaped ZnII anion binding due to second coordination sphere effects. This complex that acts as an insulator with respect to the environment nicely illustrates the critical role of the architecture surrounding (DPC vs. water). In conclusion, this study shows that both water- the coordination site of the metallo-receptor. solubilized Rim4 derivatives, WRim4 as well as its micelle II The study also gives new fundamental insights relative to the encapsulated equivalent, MRim4, behave as good ligands for Zn impact of a micellar environment: first for metal ion coordination binding in water, and the corresponding complexes as remarkable and second, for guest hosting by a metallo-receptor comprising a biomimetic receptors for small organic guests that bind as anions cavity for binding, as in enzymes. Looking at the literature, we in spite of their intrinsic hydrophilicity. It also shows that the found no data on the metal coordination aspect. However, zwitterionic DPC micelle which mimics the hydrophobic proteic examples of metallo-probes directly incorporated into micelles environment of metallo-enzyme active sites, has very little impact shows that the major effect of micelles on receptors deprived of a on metal ion affinity and guest hosting, provided the biomimetic cavity, beside their solubilization into the aqueous medium, is an site is well structured and presents a pre-shaped receptor pocket. enhanced affinity for lipophilic guests, which is assignable to the Hence, micelle incorporation of a lipophilic molecular receptor can hydrophobic effect.[24,36–39] The interesting case that we have be considered as an easy alternative to difficult synthetic work, as highlighted is that of an uranyl-salophen complex embedded in it does not disrupt its binding ability towards charged species cationic micelles[23,40] binds the highly hydrophilic fluoride anion (metal cations or anions). It represents a quite general approach with an increase of two orders of magnitude of the affinity relative that can be used with a wide variety of systems, thus opening new to the water-soluble version of the complex.[8] The electrostatic perspectives for molecular recognition in water, either for sensing, stabilization of the bound fluoride anion with the cationic micelle transport or catalysis. We are currently exploring the hydrolytic is probably a factor which explains this increased affinity. reactivity of these complexes. With receptors presenting a cavity for guest hosting, only a few Manuscript examples exploring the micellar effect were reported with resorcinarene deep cavitands.[41,42] In these cases, an Experimental Section enhancement of guest binding was also observed (up to a factor of 180 in the presence vs. in the absence of DPC micelles in General: All solvents and reagents were purchased from suppliers and water), but its enthalpic origin indicated that it is related to a used without further purification. THF was distilled over a PureSolv PS- structural effect of the micellar environment that prevents the Micro Inert Technology purification system. The NMR spectra were dimerization of the receptor in the absence of the hydrophobic recorded with four spectrometers: a Varian 600 MHz, a Bruker UltraShield guest. The case study reported here addresses a different CryoProbe 500 MHz, a Bruker Advance 500 MHz, and a Bruker ARX 250 MHz and the chemical shifts (δ) were referred to SiMe4. Mass question, which is quite general and relevant to biological spectroscopy (ESI MS) analysis were performed on a ThermoFinnigen systems: what is the impact of the micellar environment on metal LCQ Advantage spectrometer using methanol or water as solvents. IR ion affinity and anionic guest hosting? In metallo-enzymes, the spectra were recorded with a Perkin-Elmer Spectrum on FTIR active site is generally surrounded by a hydrophobic environment spectrometer equipped with a MIRacleTM single reflection horizontal ATR provided by the proteic core, whereas water-solubility is insured unit. ITC experiments were conducted on a NanoITC TA instruments by a polar surface. Here, both systems WRim4 and MRim4 calorimeter at 298 K. pD/pH measurements were performed with a Mettler- present the same rigid bowl-shape hydrophobic cavity for guest Toledo U402-M3-S7/200 long combination pH micro electrode. The hosting and the same first coordination sphere, with four indicated pD values refer to pH measured in D2O, with a correction of 0.4 pH units that allows comparison between these two notions, as previously imidazole arms preorganized on the resorcinarene core for metal [43,44] reported. Fits presented for 1:1 binding constants and pKa Accepted ion binding. This study addresses the comparison of the determination were obtained from Excel. coordination properties either in the highly polar water environment, or in the lipophilic environment provided by the DPC Cavitand 3. The tetrabromocavitand 2 (2.52 g, 1.47 mmol) was dissolved micelles. To our surprise, both systems revealed to be as efficient in dry THF and dried under vacuum for 1 hour at 80 °C three times. Under for Zn2+ binding, with very similar enthalpic and entropic argon, the reagent was then dissolved in 25 mL dry THF. At –78 °C, n- components. Likewise, the comparison of equilibria between a buthyllitium 1.6 M (11 mL, 15.4 mmol) was added slowly. After 1 hour at – free state in water for the small organic analytes (either in its 78 °C, paraformaldehyde (520 mg, 17.3 mmol) was added in one portion. The reaction medium was then agitated for 30 min at –78 °C. The flask

FULL PAPER was brought to RT, and stirred overnight. The solvent was evaporated and 32.1 (CHCH2), 27.4 ppm (CH2CH2CH2); the residue obtained was dissolved in ethyl acetate (30 mL) and a IR ν=3357, 2922, 1633, 1567, 1501, 1470, 1411, 1285, 1243, 1140, 1063, -1 + saturated solution of NH4Cl (20 mL). The organic layer was then washed 1019, 987, 747 cm ; HRMS: m/z: 1265.5743 [cavitand-H] (expected + 2+ with brine (2 x 20 mL), dried over Na2SO4 and evaporated to dryness. The 1265.5770), 1287.2 [cavitand-Na] , 633.5 [cavitand-2H] . yellow crude product obtained was purified by flash chromatography over silica (DCM/EtOH 9:1 to 8:2 v/v) to yield a white powder (1.00 g, 45 %). WRim4. Cavitand 5 (121 mg, 96 µmol) was dried three times by dissolution 1 Rf=0.12 (DCM/EtOH 9:1); H NMR (500 MHz, CDCl3, 300 K, TMS): δ=7.13 in distilled THF (2 mL) and evaporation under vacuum at 80 °C for 1 hour. 3 3 (s, 4H; Har, down), 5.90 (d, J(H,H)=6.5 Hz, 4H; Hout), 4.81 (t, J(H,H)=8.0 Hz, Under argon, the solid was dissolved in anhydrous pyridine (4.7 mL) and, 3 4H; CHCH2), 4.55 (s, 8H; Ar-CH2-O), 4.42 (d, J(H,H)=8.0 Hz, 4H; Hin), at 0 °C, mesylchloride (70 µL, 1.1 mmol) was added. The reaction medium 3 3.76 (t, J(H,H)=6.5 Hz, 8H; CH2OTIPS), 2.28 (m, 8H; CHCH2), 1.59 (m, was stirred overnight at RT, then heated at 100 °C for 24 h. A brown solid 13 8H; CH2CH2CH2), 1.07 ppm (s, 84H; TIPS); C NMR (150 MHz, CDCl3, appeared. After cooling at RT, diethylether (5 mL) was added. The solution 300 K, TMS): δ=153.8 (CAr-O), 138.3 (CAr-CH), 126.6 (CAr-CH2-OH), 120.4 was filtered and the solid washed twice with diethylether (5 mL). The crude (CAr,down), 99.9 (Cbridge), 63.2 (CH2-OTIPS), 55.8 (CH2-OH), 36.6 (CHCH2), product was dissolved in methanol (0.5 mL) and stirred for 1 hour at RT 31.2 (CH2CH2CH2), 26.4 (CHCH2), 18.3 (Si-CH-(CH3)2), 12.3 ppm (Si-CH); with an ion-exchange resin for chlorides (DOWEX). The resin was filtered IR ν=3365, 2942, 2865, 1587, 1461, 1385, 1298, 1245, 1107, 1019, 999, out and washed with methanol. The filtrate was evaporated to dryness to -1 1 962, 882, 726 cm . yield a brown solid (95 mg, 60%). H NMR (500 MHz, CD3OD, 300 K, TMS): δ=9.09 (m, 8H; pyridine), 8.52 (m, 4H; pyridine), 8.06 (m, 8H; 3 Cavitand 4. The tetraalkylated cavitand 3 (810 mg, 0.53 mmol) was firstly pyridine), 7.78 (s, 4H; Har, down), 6.97 (d, J(H,H)=1.0 Hz, 4H; Him,α), 6.79 (d, 3 3 dried by operating three times the sequential dissolution in distilled THF, J(H,H)=1.0 Hz, 4H; Him,β), 5.38 (d, J(H,H)=7.5 Hz, 4H; Hout), 4.80 (m, 8H; + 3 evaporation of the solvent under argon, with final drying under vacuum for CH2N ), 4.57 (t, J(H,H)=8,3 Hz, 4H; CH), 4.36 (s, 8H; O-CH2-Im), 4.17 (s, 3 1 hour at 80 °C. It was then dissolved in anhydrous DMF (8 mL) and added 8H; Ar-CH2-O), 4.05 (d, J(H,H)=7.5 Hz, 4H; Hin), 3.45 (s, 12H; NCH3), 2.63 13 slowly to an anhydrous DMF solution (7 mL) containing sodium hydride (m, 8H; CHCH2), 1.96 ppm (m, 8H; CH2CH2CH2); C NMR (150 MHz, solution (60% dispersed in oil, 638 mg, 15.9 mmol, washed with pentane CD3OD, 300 K, TMS) δ=155.8 (CAr-O), 147.2 (pyridine), 146.3 (pyridine), three times). After 30 min, the mixture was brought back to RT and stirred 146.1 (CAr-N), 138.9 (CAr-CH), 129.8 (pyridine), 129.6 (CAr-CAr-O), 127.5 for 2h. At 0 °C, N-methylchloromethylimidazolium hydrochloride (710 mg, (CIm,α), 124.2 (CAr,down), 124.0 (CIm,β), 101.4 (Cbridge), 64.8 (O-CH2-CAr-N), + 4.24 mmol) was added in four portions every 15 min. After 15 min at 0 °C, 63.2 (CH2N , CAr-CH2-O), 38.4 (CH), 33.4 (NCH3), 30.9 (CHCH2), 28.1 the solution was warmed up to RT and stirred overnight under argon. ppm (CH2CH2CH2); IR ν=3393, 2946, 1635, 1590, 1476, 1405, 1323, 1285, Water (80 mL) was then added and the precipitate formed was filtered off 1246, 1150, 1064, 1017, 966, 774, 686; HRMS: m/z: 378.1877 [cavitand]4+ and washed with water (2 x 20 mL). The crude product (1.02 g) obtained (expected: 378.1818). was used without further purification in the following step. 1H NMR (500 3 3 δ down MHz, CDCl , 300 K, TMS): =7.05 (s, 4H; Ar–H ), 6.93 (d, J(H,H)=1.1 [WRim4Zn]Cl4(NO3)2. The zinc complex was generated in situ in D2O by 3 3 Im,β Im,α 1 Hz, 4H; H ), 6.88 (d, J(H,H)=1.1 Hz, 4H; H ), 5.57 (d, J(H,H)=7.0 Hz, addition of one equivalent of Zn(NO3)2 to a WRim4 solution. H NMR (500 3 4H; O–CH2–O), 4.73 (t, J(H,H)=8.5 Hz, 4H; –CH–), 4.56 (s, 8H; O–CH2– MHz, D2O, pD = 7.31, 300 K, TMS): δ=8.91 (m, 8H; pyridine), 8.60 (m, 4H; 3 Im), 4.20 (s, 8H; Ar–CH2–O), 4.16 (d, J(H,H)=7.0 Hz, 4H; O–CH2–O), 3.72 pyridine), 8.10 (pyridine, m, 8H), 7.59 (s, 4H; Har, down), 7.20 (s, 4H; Him,β), 3 3 (t, J(H,H)=6.5 Hz, 8H; CH2–OTIPS), 3.61 (s, 12H; N–CH3), 2.22 (m, 8H; 6.65 (bs, 4H; Him,α), 5.62 (d, J(H,H)=7.0 Hz, 4H; Hout), 4.70 (s, 8H; O-CH2- 13 CH–CH2), 1.55 (m, 8H; CH2–CH2–OTIPS), 1.06 ppm (m, 84H; TIPS); C Im), 4.49 (s, 8H; Ar-CH2-O), 4.28 (bs, 4H; Hin), 3.76 (bs, 12H; NCH3), 2.55 Manuscript δ 13 NMR (150 MHz, CDCl3, 300 K, TMS): =154.0 (CAr–O), 144.4 (CAr–N), (m, 8H; CHCH2), 2.12 ppm (m, 8H; CH2CH2CH2); C NMR (150 MHz, D2O, 137.8 (CAr–CH), 127.5 (CHIm,β), 123.6 (CAr–CH2–O), 122.1 (CHIm,α), 120.6 pD = 7.31, 300 K, TMS): δ=153.6 (CAr-O), 145.8 (pyridine), 145.1 (CAr-N), (CHAr,down), 99.4 (O–CH2–O), 64.7 (O– CH2–Im), 63.0 (CH2–OTIPS), 61.9 144.1 (pyridine), 137.0 (CAr- CHar,down ), 128.4 (pyridine), 125.3 (Cim,α), (Ar–CH2–O), 36.5 (CH), 32.9 (CH2–CH2–OTIPS), 31.0 (N–CH3), 26.2 124.0 (Cim,β), 122.8 (CAr-CAr-O), 122.4 (CHAr,down), 100.0 (Cbridge), 62.5 (O- ν + (CH–CH2), 18.1 (Si–C–CH3), 12.1 ppm (Si–C); IR =3357, 2942, 2866, CH2-Im), 61.7 (Ar-CH2-O), 61.5 (CH2N ), 36.3 (CH), 33.3 (NCH3), 28.9 1592, 1500, 1464, 1389, 1285, 1245, 1149, 1106, 1067, 1016, 969, 882, (CHCH2), 26.1 ppm (CHCH2CH2); IR ν=3430, 2935, 1642, 1563, 1349, -1 745, 681 cm . 1245, 1152, 1048, 1024, 994, 968, 829, 765 cm-1.

II Cavitand 5. The tetraimidazole cavitand 4 (976 mg, 0.52 mmol) was dried [MRim4Zn ](ClO4)2. From MRim4: The ligand Rim4 (1.04 mg, 0.64 µmol), under vacuum at 60 °C for 1 h. Under argon, it was dissolved in THF/H2O synthesized as previously reported,[20] was suspended in an acidic solution 1:1 v/v (54 mL). Trifluoroacetic acid (7 mL) was then added slowly. The of DPC (10.7 mg, 20 mM, pD = 2) in D2O (for NMR studies, H2O for ITC medium was stirred overnight at RT. The solvent and TFA were studies). The suspension was stirred at RT for 3 hours, until a clear evaporated. Toluene (3 mL) was added and evaporated for better water solution was obtained, and used as such for NMR or ITC studies. The

elimination (three times). The residue was dissolved in methanol (12 mL) associated zinc(II) complex MRim4Zn was generated by addition of one - and treated with a DOWEX resin conditioned for HO exchange. After 1 h, equivalent of Zn(ClO4)2. Direct synthesis of MRim4Zn: The complex the resin was filtered off and washed with methanol. The filtrate was [Rim4Zn(EtOH)](ClO4)2 (1.2 mg, 0.7 µmol), prepared as previously evaporated to dryness. The yellowish solid obtained was purified by flash reported,[20] was suspended in an HEPES buffer (1.47 mL, 50 mM, pD = chromatography over silica (MeOH/DCM 1:9 to 2:8 v/v, 0.3 % NH3) to yield 7.4) containing DPC (10.3 mg, 20 mM), stirred at RT overnight then heated a yellow oil (627 mg, 93 % over two steps). Rf=0.43 (MeOH/DCM 15:85, to 50 °C for 30 min. The limpid solution obtained was used as such for 1 0.3 % NH3); H NMR (500 MHz, CD3OD, 300 K, TMS): δ=7.33 (s, 4H; Har, NMR studies. Accepted 3 3 down); 7.31 (d, J(H,H)=1.4 Hz, 4H; Him); 7.21 (d, J(H,H)=1.4 Hz, 4H; Him), 3 3 5.71 (d, J(H,H)=7.0 Hz, 4H; Hout), 4.69 (t, J(H,H)=6.4 Hz, 4H; CH), 4.67 . Typically, (ca. 0.8 mg, 1 mM) 3 General NMR titration procedure WRim4 (s, 8H; O-CH2-Im), 4.40 (s, 8H; Ar-CH2-O), 4.17 (d, J(H,H)=7.0 Hz, 4H; or Zn (0.5 mM, DPC 20 mM) was dissolved in 500 µL buffered D2O. 3 MRim4 Hin), 3.69 (s, 12H; NCH3), 3.63 (t, J(H,H)=6.5 Hz, 8H; CH2OH), 2.32 (m, The buffer was HEPES 100 mM (50 mM for micelles), brought to pD = 7.4 13 8H; CHCH2), 1.47 ppm (m, 8H; CH2CH2CH2); C NMR (150 MHz, CD3OD, with a NaOH 1 M solution. 5 µL (1 equivalent) of a Zn(NO3)2 0.1 M solution 300 K, TMS): δ=155.7 (CAr-O), 145.6 (CAr-N), 139.4 (CAr-CH), 125.2 (CAr- in D2O were added when WRim4 was used. Aliquots (between 2 and 20 CAr-O), 124.6 (CIm,α), 123.4 (CAr,down), 121.5 (CIm,β), 101.4 (Cbridge), 64.3 µL) of a solution of guest (0.05 M, 10 µL for 1 equivalent) in the same buffer (CAr-CH2-O), 62.8 (OCH2Im), 62.6 (CH2OH), 38.3 (CHCH2), 34.7 (NCH3), were added progressively until saturation and 1H NMR spectra were

FULL PAPER recorded after each addition. The final pD of the solution was measured to [14] O. Seneque, M.-N. Rager, M. Giorgi, O. Reinaud, J. Am. Chem. Soc. ensure it was constant during the whole experiment. 2000, 122, 6183–6189. [15] D. Coquière, S. Le Gac, U. Darbost, O. Sénèque, I. Jabin, O. Reinaud, Org. Biomol. Chem. 2009, 7, 2485-2500. General ITC titration procedure. 1 mL of host solution was introduced [16] J.-N. Rebilly, B. Colasson, O. Bistri, D. Over, O. Reinaud, Chem. Soc. into the cell of the calorimeter. The titrant solution was added via a 250 µL Rev. 2015, 44, 467–489. [17] N. Le Poul, Y. Le Mest, I. Jabin, O. Reinaud, Acc. Chem. Res. 2015, syringe in 24 injections of 10 µL with a 400 s interval. A blank was recorded 48, 2097–2106. by replacing the solution in the cell by 1 mL solvent. The obtained data [18] G. Izzet, X. Zeng, H. Akdas, J. Marrot, O. Reinaud, Chem Commun was processed with Nanoanalyze software. 2007, 810–812. [19] J. Gout, S. Rat, O. Bistri, O. Reinaud, Eur. J. Inorg. Chem. 2014, 2819–2828. [20] A. Parrot, S. Collin, G. Bruylants, O. Reinaud, Chem. Sci. 2018, 9, 5479–5487. [21] O. Bistri, B. Colasson, O. Reinaud, Chem. Sci. 2012, 3, 811–818. [22] A. Inthasot, N. Le Poul, M. Luhmer, B. Colasson, I. Jabin, O. Reinaud, Inorg. Chem. 2018, 57, 3646–3655. [23] M. Cametti, A. Dalla Cort, K. Bartik, ChemPhysChem 2008, 9, 2168– Acknowledgements 2171. [24] F. Su, R. Alam, Q. Mei, Y. Tian, C. Youngbull, R. H. Johnson, D. R. Meldrum, PLoS ONE 2012, 7, e33390. This project was supported by the CNRS and the Ministère de l’ [25] E. Brunetti, A. Inthasot, F. Keymeulen, O. Reinaud, I. Jabin, K. Bartik, Enseignement Supérieur et de la Recherche. The authors also Org. Biomol. Chem. 2015, 13, 2931–2938. [26] A. Višnjevac, J. Gout, N. Ingert, O. Bistri, O. Reinaud, Org. Lett. 2010, acknowledge the FRIA-FRS (E. B PhD grant), the FNRS and the 12, 2044–2047. Van Buuren foundation for founding of the ITC equipment. [27] S. Rat, J. Gout, O. Bistri, O. Reinaud, Org. Biomol. Chem. 2015, 13, 3194–3197. [28] K.-D. Zhang, D. Ajami, J. Rebek, J. Am. Chem. Soc. 2013, 135, Keywords: biomimetism • water-soluble • micelles • 18064–18066. [29] B. C. Gibb, R. G. Chapman, J. C. Sherman, J. Org. Chem. 1996, 61, resorcinarene • zinc 1505–1509. [30] U. Darbost, M.-N. Rager, S. Petit, I. Jabin, O. Reinaud, J. Am. Chem.

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A well-structured biomimetic core Solène Collin, Arnaud Parrot, Lionel capable of ZnII coordination and Marcelis, Emilio Brunetti, Ivan Jabin, anion hosting is efficiently Gilles Bruylants, Kristin Bartik, and transferred into water via two Olivia Reinaud* strategies and it retains its remarkable properties. Page No. – Page No. ((Insert TOC Graphic here: max. width: 5.5 cm; max. height: 5.0 cm)) Submerging a Biomimetic Metallo- Receptor in Water for Molecular Recognition: Micellar Incorporation or Water Solubilization? A Case Study

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