A Water-Soluble Leggero Pillar[5]arene

The study of aqueous-phase molecular recognition of artificial receptors is one of the frontiers in supramolecular chemistry since most biochemical processes and reactions take place in an aqueous medium and heavily rely on it. In this work, a water-soluble version of leggero pillar[5]arene bearing eight positively charged pyridinium moieties (CWP[5]L) was designed and synthesized, which exhibited good binding affinities with certain aliphatic sulfonate species in aqueous solutions. Significantly, control experiments demonstrate that the guest binding performance of CWP[5]L is superior to its counterpart water-soluble macrocyclic receptor in traditional pillararenes.

Since most biochemical reactions take place in water, the investigation of molecular recognition in aqueous phases is of greater research value than in organic phases. In the past few years, cationic water-soluble macrocyclic receptors have received tremendous attention in supramolecular community due to their great potential in complexing with important organic anionic species, and many valuable applications such as antibiotics [44], metabolism regulation [45], selective precipitation [33], self-assembly [46] and supra-amphiphiles [47] have been explored on the basis of this feature. Thus, synthesis of novel cationic watersoluble macrocyclic receptors and investigating their host-guest properties in aqueous phase are highly significant and worth exploring.
amphiphiles [47] have been explored on the basis of this feature. Thus, synthesis of novel cationic water-soluble macrocyclic receptors and investigating their host-guest properties in aqueous phase are highly significant and worth exploring.
In this work, the first cationic water-soluble version of the leggero pillar [5]arene (i.e., CWP[5]L) is designed and successfully synthesized. Four aliphatic sulfonate guests with different chain lengths (G1-G4) are selected to investigate the recognition properties of CWP [5]L in aqueous media (Scheme 1). Control experiments employing its counterpart macrocyclic derivative CWP [5]A from traditional pillararenes confirm that CWP [5]L has better binding performance owing to its superior structural flexibility.

Results
As shown in Scheme 1, CWP[5]L and CWP [5]A bearing eight and ten cationic pyridinium moieties could be quantitatively prepared through a SN2 nucleophilic substitution by reacting their corresponding perbromoethylated macrocyclic derivatives BrP [5]L and BrP [5]A with pyridine (also as solvent), respectively, and the target receptors were fully characterized by 1 H/ 13 C NMR and high-resolution mass spectroscopy (HRMS) ( Figures  S1-S6).
The binding behaviors of CWP[5]L with G1-G4 were first investigated by 1 H NMR spectroscopy. As shown in Figure 1a, when we mixed CWP[5]L (4.0 mM) and 1.0 equiv. of G1 in D2O, the 1 H NMR spectrum displayed only one set of resonance signals distinct from those of the single component host and guest, indicating that the binding complex was formed, and the complexation between CWP[5]L and G1 was a fast exchange process on the NMR timescale. Compared with the free guest, protons Ha-Hc of G1 showed remarkable upfield shifts and broadening effects as a result of the inclusion-induced shielding effects, while protons Hd on the terminal methyl showed downfield shifts due to the deshielding effect. These changes indicated that G1 was threaded into the host cavity, forming an interpenetrated inclusion complex. On the other hand, the host was deshielded by the included guest, and the proton signals of CWP [5]L derived from the

Results
As shown in Scheme 1, CWP[5]L and CWP [5]A bearing eight and ten cationic pyridinium moieties could be quantitatively prepared through a S N 2 nucleophilic substitution by reacting their corresponding perbromoethylated macrocyclic derivatives BrP [5]L and BrP [5]A with pyridine (also as solvent), respectively, and the target receptors were fully characterized by 1 H/ 13 C NMR and high-resolution mass spectroscopy (HRMS) (Figures S1-S6).
The binding behaviors of CWP[5]L with G1-G4 were first investigated by 1 H NMR spectroscopy. As shown in Figure 1a, when we mixed CWP[5]L (4.0 mM) and 1.0 equiv. of G1 in D 2 O, the 1 H NMR spectrum displayed only one set of resonance signals distinct from those of the single component host and guest, indicating that the binding complex was formed, and the complexation between CWP[5]L and G1 was a fast exchange process on the NMR timescale. Compared with the free guest, protons H a -H c of G1 showed remarkable upfield shifts and broadening effects as a result of the inclusion-induced shielding effects, while protons H d on the terminal methyl showed downfield shifts due to the deshielding effect. These changes indicated that G1 was threaded into the host cavity, forming an interpenetrated inclusion complex. On the other hand, the host was deshielded by the included guest, and the proton signals of CWP [5]L derived from the pyridinium moieties (H 1 ) and substituted phenylene subunits (H 2 and H 3 ) exhibited downfield displacement. pyridinium moieties (H1) and substituted phenylene subunits (H2 and H3) exhibited downfield displacement. The host-guest binding behavior between CWP[5]L and G1 was also investigated by 2D ROESY analysis. As shown in Figure 1b, the correlation signals between the alkyl chain protons (Hc) of G1 and the phenylene and pyridinium protons (H1 and H2) of CWP[5]L were clearly observed, further confirming the interpenetrated geometry. Besides, similar complexation-induced shielding/deshielding effects were also observed in the mixtures of CWP [5]L and other selected sulfonate guests (G2-G4), respectively, indicating that these host-guest assemblies have a similar binding mode, i.e., the macrocyclic cavity of CWP [5]L is threaded by the alkyl chain of the guests. Interestingly, it should be noted that with the decrease in the alkyl chain length from G1 to G4, the resonance signals for the terminal methyl protons (Hd) experienced a shift from downfield (G1 and G2) to upfield The host-guest binding behavior between CWP[5]L and G1 was also investigated by 2D ROESY analysis. As shown in Figure 1b, the correlation signals between the alkyl chain protons (H c ) of G1 and the phenylene and pyridinium protons (H 1 and H 2 ) of CWP[5]L were clearly observed, further confirming the interpenetrated geometry. Besides, similar complexation-induced shielding/deshielding effects were also observed in the mixtures of CWP [5]L and other selected sulfonate guests (G2-G4), respectively, indicating that these host-guest assemblies have a similar binding mode, i.e., the macrocyclic cavity of CWP [5]L is threaded by the alkyl chain of the guests. Interestingly, it should be noted that with the decrease in the alkyl chain length from G1 to G4, the resonance signals for the terminal methyl protons (H d ) experienced a shift from downfield (G1 and G2) to upfield (G3 and G4) upon mixing with CWP[5]L (Figure 1a and Figures S7-S9), suggesting that the terminal groups of G1 and G2 are protruded out of the macrocyclic cavity and the corresponding inclusion complexes could also be considered as [2]pseudorotaxane structures, respectively ( Figure 2).
(G3 and G4) upon mixing with CWP[5]L (Figures 1a and S7-S9), suggesting that the terminal groups of G1 and G2 are protruded out of the macrocyclic cavity and the corresponding inclusion complexes could also be considered as [2]pseudorotaxane structures, respectively ( Figure 2).  To quantitatively estimate the interactions between CWP[5]L and the sulfonate guests, 1 H NMR titration experiments were further implemented to afford the association constants (K a ) for the host-guest inclusion complexation in D 2 O (Figures S10-S13). As shown in Figure 3a-d, CWP[5]L has good binding affinities towards all the guests, and the K a values for 1:1 complexation with G1-G4 were determined to be 50,500, 34,300, 11,600 and 9330 M −1 , respectively. Interestingly, a marked increase in the K a values was observed in the order of G4 < G3 < G2 < G1, which could be attributed to the enhanced hydrophobic interaction and the guests' increasing chain length.  Figures 1a and S7-S9), suggesting that the terminal groups of G1 and G2 are protruded out of the macrocyclic cavity and the corresponding inclusion complexes could also be considered as [2]pseudorotaxane structures, respectively ( Figure 2). To quantitatively estimate the interactions between CWP[5]L and the sulfonate guests, 1 H NMR titration experiments were further implemented to afford the association constants (Ka) for the host-guest inclusion complexation in D2O (Figures S10-S13). As shown in Figure 3a-d, CWP [5]L has good binding affinities towards all the guests, and the Ka values for 1:1 complexation with G1-G4 were determined to be 50,500, 34,300, 11,600 and 9330 M −1 , respectively. Interestingly, a marked increase in the Ka values was observed in the order of G4< G3 < G2 <G1, which could be attributed to the enhanced hydrophobic interaction and the guests' increasing chain length. Given that CWP[5]L exhibited strong binding affinities with linear sulfonate guests, we were curious to find some differences in the binding performance between CWP[5]L and its counterpart water-soluble macrocyclic derivative CWP [5]A from traditional pillararenes. Subsequently, controlled 1  Given that CWP[5]L exhibited strong binding affinities with linear sulfonate guests, we were curious to find some differences in the binding performance between CWP[5]L and its counterpart water-soluble macrocyclic derivative CWP [5]A from traditional pillararenes. Subsequently, controlled 1 H NMR titration experiments by using CWP [5]A were carried out ( Figures S14-S17), and the K a values for G1-G4 were determined to be 39,500, 9760, 6210 and 5900 M −1 , respectively (Figure 3e-h). Analogously, the K a values increase in the order of G4 < G3 < G2 < G1, again confirming the pivotal role of hydrophobic interaction in modulating the host-guest inclusion complexation in the aqueous phase. Significantly, the binding constants of CWP [5]L are larger than those of its counterpart CWP [5]A, indicating that the de-functionalized and free-rotation phenylene units invest CWP [5]L with more superior cavity adaptability and relatively low in-cavity electron density for binding anionic species.

General Information
Starting reagents and solvents were commercially available and used without further purification unless stated otherwise. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AVANCE III 400 MHz instrument at room temperature. The 2D ROESY NMR spectra were recorded on a Bruker AVANCE III 600 MHz instrument. Mass spectra were recorded on a Bruker Agilent1290-micrOTOF Q II High-resolution (HR) mass spectrometry instrument.

Synthesis of CWP[5]L
A pyridine (3 mL) solution of BrP [5]L (0.6 g, 0.4 mmol) was heated at 100 • C for 10 h. Then, the resulting precipitate was filtered and washed with CH 2 Cl 2 to afford the target compound as a light yellow solid (0.82 g, 95%). 1

Determination of Association Constants
To determine the association constant, 1 H NMR titrations using a nonlinear leastsquares curve-fitting method were performed at 298 K in D 2 O. The association constants (K a ) were obtained for each host-guest combination from the following equation [48]:

Conclusions
In conclusion, we successfully synthesized the first water-soluble derivative of leggero pillar [5]arene (i.e., CWP[5]L), where the presence of eight cationic pyridinium moieties makes it a highly effective receptor for anionic sulfonate species in aqueous solutions. NMR experiments demonstrated that all the guests could be encapsulated into the cavity of CWP[5]L to form 1:1 host-guest complexes with interpenetrated geometry, and the binding affinities could be remarkably enhanced by increasing the alkyl chain length of the guests due to the growing hydrophobic interaction. More importantly, controlled experiments employing its counterpart water-soluble macrocyclic receptor derived from traditional pillararenes confirmed that CWP[5]L had better binding performance because of its superior structural flexibility and cavity adaptability originating from the free-rotation phenylene subunit. Above all, given the superior binding performance toward anionic species in water, we firmly believe that CWP[5]L will find far-ranging applications in, for example, molecular recognition, environmental remediation, biomedicine, material science and other related fields.