**3. Results**

Three octyl-benzoureido-15-crown-5-ethers have been prepared for the studies described here. The octyl-isocyanate ( *<sup>R</sup>*)-(−)-2-octyl isocyanate, (*S*)-(+)-2-octyl isocyanate, 2-ethylhexyl isocyanate were treated with the corresponding 4-aminobenzo-15-crown-5-ether (CHCl3, 65 ◦C, overnight) to a fford after precipitation from hexane **1**, **r2**, **s2** and **3**, respectively, as powders (Scheme S1). 1H-, 13C-NMR, ESI-MS, and HR-MS spectroscopic data are in accord with proposed structures (see Supplementary Materials for details, Figures S1–S8).

The generation of functional transporting superstructures of **1**, **r2**, **s2** and **3** is based on three encoded features: (1) they contain macrocyclic cation-binding moieties [3–8]; (2) the supramolecular guiding interaction is the urea head-to-tail H-bond association, reminiscent with the amide H-bonding moiety in protein [9–15]; (3) the alkyl tails control the H-bonding self-assembly and induce variable hydrophobic stabilization at the interface with the bilayer membrane.

1H NMR dilutions experiments on solutions in CDCl3 of **1**, **r2**, **s2**, **3** show a variable downfield shift of both NH protons upon increasing the concentration, which is indicative of self-association through intermolecular H-bonding (Figure 2, Figures S9–S11). To quantify the degree of the aggregation, the NH shifts at different concentrations were fitted to the NMR CoEK aggregation model (Nelder–Mead method) using Bindfit [9]. The determined values of the aggregation constants, as well as the cooperativity factors, are given in Table 1. There is encouraging strong cooperative H-bonding effect. The most associated compound **1** contains the linear *n*-octyl sidearm while the formation of aggregates is hindered when crowded supplementary residues block the H-bonding and slightly deactivate (~25% decrease) the self-association in **r2**, **s2** and **3**.

**Figure 2.** The difference in chemical shifts of the urea-type Ha protons relative to chemical shifts δ at 0.01 M plotted against the concentration of intermolecular H-bonded oligomers **1**, **r2**, and **3**, in CDCl3 at 25 ◦C.

**Table 1.** Association Constants Ke for Numerical Fits a Monomer/Dimer/Aggregate model, obtained from NMR dilution experiments in CDCl3 at 25 ◦C [9,13].


a The factor ρ = Kd/Ke highlight the cooperativity of aggregation with ρ < 1 positive cooperativity, ρ = 1, no cooperativity and ρ > 1 negative cooperativity for Ke aggregation vs. Kd dimerization.

The ion-transport activities were evaluated by the HPTS assay [24,25]. <sup>l</sup>-<sup>α</sup>-phosphatidylcholine, EYPC liposomes (Large Unilamellar Vesicles-LUV, 100 nm) were filled with a pH-sensitive dye, HPTS and 100 mM NaCl in a phosphate buffer (10 mM, pH 6.4). The liposomes were then suspended in an external phosphate buffer (10 mM, pH 6.4) containing 100 mM of MCl, M<sup>+</sup> = Na<sup>+</sup>, K<sup>+</sup>. Then, after addition of **1**, **r2**, **s2**, **3** in the bilayer membrane via injection of 20 μL of compound aliquots from stock 10 mM Dimethylsulfoxide (DMSO) solutions, see the final concentration values (% mol of the compound/mol of lipid) in hill plot analyses in Figures S12–S25), an external pH gradient was created by addition of NaOH. The internal pH change inside the liposome was monitored by the change in the fluorescence of HPTS (Figure 3a,b) [26].

*Chemistry* **2020**, *2*

**Figure 3.** Schematic representation of the translocation mechanism in the presence of macrocycles (**a**) without or (**b**) with FCCP proton transporter. (**c**) Normalized I460/I403 (I/I0) versus time profiles for the transport of K<sup>+</sup> or Na<sup>+</sup> across the bilayer membrane promoted by **r2** or **s2** (32.3 mol%).

Compounds **1**,**r2**, **s2**, and **3** are presenting subtle variations on the transport activities of monovalent K<sup>+</sup> and Na<sup>+</sup> cations, depending the structure of isomeric octyl substituents of ureido-benzo-15-crown-5 macrocycle. We note that enantiomers **s2** and **r2** have rather same activity for translocation of K<sup>+</sup> and Na<sup>+</sup> at the same concentration (Figure 3c). Under the same conditions, compounds **1**, **r2**, **s2** and **3** (12.9 mol%) present one order of magnitude higher initial transport rate for K<sup>+</sup> cations than for Na<sup>+</sup>, with kinetic selectivity of SK+/Na+ = 3 to 6.6, depending on the compound (Tables S1 and S2 and Figure 4a).

**Figure 4.** Bar graphs showing (**a**) the pseudo-first-order rate constants *k* (s−1) for the transport of K<sup>+</sup> and Na<sup>+</sup> cations and (**b**) rate enhancement and K+/Na<sup>+</sup> selectivity, for the macrocyclic carriers **1**, **s2**, **3**, (12.9 mol%) in presence or absence of protogenic FCCP carrier.

Hill analysis [27] (Table 2, Figures S12–S25) revealed all macrocycles present K<sup>+</sup> over Na<sup>+</sup> selectivity, which is consistent with previous results. [8,15] Compound **1** is the most active, as it has the lowest EC50 for both Na<sup>+</sup> (10.6 mol%), and K<sup>+</sup> (4.1 mol%), much lower than its isomers following the transport activity sequence of **1** > **s2** > **3** within the μM concentration range. Compared with Valinomicin (EC50K+ = 5 pM) they are several orders of magnitude for K<sup>+</sup> activity. All the Hill coefficients are above 2, indicative of a formation of self-assembled aggregates containing more than two molecules of macrocycles, which transport cooperatively the cations, contrarily to Valinomycin acting as a carrier with a Hill number = 1 [27].


**Table 2.** Hill analysis results of K<sup>+</sup> and Na<sup>+</sup> transport with crown ethers **1**, **r2**, **s2**, **3**, in the presence or absence of FCCP. EC50 values expressed as mol% (% molar of compound/lipid needed to obtain 50% ion transport activity) and *n* is the Hill coefficient.

a EC50 was determined by the Hill plot, using the fractional activities at 340 s–290 s after the addition of the compound; b Hill coefficient.

According to our previous results, the addition of alkyl-benzoureido-15-crown-5-ether channels in the membrane generate a quite stable transmembrane potential, following a K<sup>+</sup> > H<sup>+</sup> electrogenic antiport through the lipid bilayer [15]. We also know that the simultaneous addition of macrocycles and FCCP leads to the electrical coupling of fluxes and the transport can proceed without a potential across the membrane via a non-electrogenic exchange [28,29].

As can be seen from Figure 4, the electrogenic component of the macrocyclic compound-mediated cation influx is stimulated by the addition of 0.1 mol% FCCP proton carrier as well, which overcome the proton transport rate-limiting barrier with a special emphasis for the transport of K<sup>+</sup> cations for which the transport rate show a 12 to 27 fold increase. This result is valid for all studied macrocycles **1**, **r2**, **s2**, **3**, for which the fractional activities Y for K<sup>+</sup> cations are also higher. This is also obvious from the EC50 values, which are strongly improving for K<sup>+</sup> but not for Na<sup>+</sup>. FCCP improves transport efficiency, but more impressively, we observe an increase of SK+/Na<sup>+</sup> selectivity from 6.6 to 45.5 in the case of **2** and from 3.6 to 48.8 in the case of **3**.

For all compounds, FCCP did improve transport activity towards Na<sup>+</sup>, although not as much as K<sup>+</sup>, and the rate of transport follows the sequence: K<sup>+</sup> > Na<sup>+</sup> > H<sup>+</sup>. As far as we know, simple artificial systems presenting so high K+/Na<sup>+</sup> selectivity are rare. It can be explained by several reasons: (i) The fact that these macrocycles can achieve this type of stable ionic potential, suited to drive protonic gradients, is simply due to their high selectivity for K<sup>+</sup>. We know from our previous studies that similar compounds are selective for the transport of K<sup>+</sup> cations, even in the presence of Na<sup>+</sup> cations. [15] (ii) the formation of complexes is highly controlled via the formation of carrier dimers (benzo-15-crown-5-ether2K+) or higher oligomeric channels (benzo-15-crown-5-ethernK+). [13] Log *P* = 3.63−3.66 values, which are often used for evaluating the lipophilicity of the compound, are quite similar for all studied compounds **1**, **r2**, **s2**, **3**, thus, their partitions within the membrane might be similar (Table 3). Within this context, the branched alkyl chains might have a negative impact on the formation of the aggregates inside the lipid bilayer. Branched alkyl tails may favor the presence of low dimensional self-assembles species of **r2**, **s2**, or **3**. They show lower permeability than **1** (Figure 4a), but they lead to an increased SK+/Na<sup>+</sup> selectivity via proton-mediated electrogenic conductance across the membrane (Figure 4b).


3.63

**3**

**Table 3.** Calculated logP (cLogP) values were obtained using VCC labs online calculator ALOGPS 2.1 to assess the lipophilicity of thecompounds[30].
