Calix[6]arene-based atropoisomeric pseudo[2]rotaxanes

Some examples of atropoisomeric pseudorotaxanes in which the isomerism arises by the different conformations adopted by the wheel are reported here. Upon threading hexahexyloxycalix[6]arene 1 with ammonium axles 2+ or 3+, bearing biphenyl or trifluoromethylbenzyl moieties, respectively, two atropoisomeric pseudorotaxanes were formed in which the calix[6]-wheel 1 adopts the 1,2,3-alternate and cone conformations. The interconversion between them cannot be obtained by simple rotation around the ArCH2Ar bonds of the calixarene wheel, which is blocked by the presence of the axle inside its cavity. Therefore, it can only be obtained through a mechanism of de-threading/re-threading of the axle. In all the examined cases, the 1,2,3-alternate and cone atropoisomers are, respectively, the kinetic and the thermodynamic ones.

An amazing aspect of rotaxanes and catenanes is their ability to adopt novel forms of isomerism. More in detail, (pseudo)rotaxane or catenane architectures can show novel stereoisomeric forms as a result of the "social" [14] relationship between their components.
Recently, Goldup's group assembled a mechanically planar chiral rotaxane [15,16] (I and I*, Figure 1) consisting of achiral components. The combination of a macrocycle with rotational asymmetry and a directional thread with non-equivalent ends is the cause of chirality in this example ( Figure 1). Interestingly, our group showed that a chiral pseudorotaxane can be generated upon threading a tertiary ammonium axles in a directional (non-flat) calixarene-wheel (II and II*, Figure 1) [17]. In this case the chirality is created by the directionality of the calixarene wheel in a cone conformation, which differentiates the two alkyl chains around the prochiral ammonium center.  [15,16] (I and I*) and of the chiral pseudorotaxane (II and II*) reported by our group [17].
In 2010, for the first time, an example of sequence isomerism was reported by Leigh's group [18], caused by two different flat wheels that can be located differently along a directional thread III and IV ( Figure 2). As an evolution of this concept, we envisaged a sequence stereoisomerism if two directional nonflat wheels, such as calixarenes or cyclodextrins, are threaded along an axle to give a pseudo [3]rotaxane architecture V-VII (Figure 2), where three sequential stereoisomers can arise. We showed that this stereoisomerism can be effectively controlled when two calix [6]arene wheels are threaded along a bis(benzylalkylammonium) axle [19], where the stereoselective formation of the pseudo [3]rotaxane with endo-alkyl orientation VIII was observed [19].
pair of calixarene ArCH 2 Ar methylene protons. These can show diasterotopicity resulting in AX or AB systems. Specifically, a 1 H NMR methylene proton Δδ value of at least 0.7 shows that the two respective proximal aromatic rings are oriented syn, as in the cone conformation. In contrast, a Δδ value of 0.3 or less is attributable to an anti-orientation between the phenol rings, as in alternate conformations. The de Mendoza's " 13 C NMR single rule" [30,31], is focused on the 13 C NMR chemical shift of the ArCH 2 Ar methylene C, which is 30-33 ppm for the syn-orientation of the proximal phenol rings and typically 36-39 ppm with anti-positioned phenol rings as in alternate conformations.
The threading of calix [6]arene macrocycles in conformations different than the cone one has been rarely observed [17]. Interestingly, the assembling of interpenetrated structures in which the wheel adopts different conformational isomers, could pave the way to mechanomolecules which exhibit novel isomeric forms.
Prompted by these considerations, some examples of pseudorotaxane isomers in which the isomerism arises by the different conformations adopted by the calixarene wheel are reported here.

Results and Discussion
With this goal in mind, we conducted an initial screening in order to select the ammonium axles and the calix [6]arene-wheel most suitable for our purposes. At the end of our screening, we focused our attention on hexahexyloxycalix [6]arene 1 as the wheel and bis(4-biphenylmethyl)ammonium (2 + ) and bis(4-trifluoromethylbenzyl)ammonium (3 + , TFPBsalts) as the threads. The synthetic pathway to 2 + ·TFPB − and 3 + ·TFPB − salts is outlined in Scheme 1, while calix [6]arene 1 was obtained following a known procedure [40].
The 1 H NMR spectrum of hexahexyloxycalix [6]arene 1 in CDCl 3 at 298 K shows broad ArCH 2 Ar signals indicative of a conformational mobility of the macrocycle in which the inversion between the calix [6]arene conformations ( Figure 5), occurs by means of rotation around the ArCH 2 Ar bonds.
By lowering the temperature, the ArCH 2 Ar signal decoalesced to form a single AX system (3.34/4.49 ppm) and one broad singlet (3.77 ppm). This pattern is only compatible with the presence of a 1,2,3-alternate conformation of calix [6]arene 1 ( Figure 5). This was confirmed by a 2D HSQC spectrum of 1 at 233 K which evidenced the presence of ArCH 2 Ar correlations between the AX system at 3.34/4.49 ppm with a carbon resonance at 29.4 ppm, related to syn-oriented Ar rings [29]. Diagnostic of the presence of the 1,2,3-alternate conformation of 1 is the presence of the broad singlet at 3.71 ppm which correlates in the HSQC spectrum with a carbon resonance at 34.1 ppm [30], related to anti-oriented Ar rings. A close inspection of the 1D and 2D NMR spectrum of 1 in CDCl 3 at 233 K clearly evidenced the presence of a less abundant conformer of 1. The nature of this minor conformer can be inferred by the work of Reinhoudt and co-workers which showed [41] that the conformations preferentially adopted by calix [6]arene hexa-ethers are the cone and 1,2,3-alternate ones. In accordance, 2D COSY and HSQC spectra of 1 at 233 K clarified that this minor conformer was the cone one through the presence of an AX system at 3.35/4.42 ppm (COSY), which correlates with a carbon resonance at 29.1 ppm (HSQC), related to syn-oriented Ar rings (cone conformation). The coalescence temperature of the methylene protons was ascertained at 328 K in CDCl 3 ; below this temperature the conformations of 1 were frozen, while at temperatures above 328 K the conformational interconversion is fast with respect to the NMR time scale (400 MHz). From the coalescence data we calculated a barrier of 14.6 kcal/mol for this process. In summary, the VT 1 H NMR studies indicate that the 1,2,3-alternate is the most stable conformation for hexahexyloxycalix [6]arene 1 in solution. This conclusion is in perfect accord with the results previously reported by Reinhoudt [41], which evidenced an increased stabilization of the 1,2,3-alternate conformation of calix [6]arenes when the alkyl substituents at the lower rim are increased in size [41].
As expected [40], no evidence of interaction between 2 + and 1 was detected by NMR, when 2 + was added as its chloride salt to a CDCl 3 solution of 1. However, when 2 + was added as its TFPB − salt to a CDCl 3 solution of 1, then dramatic changes were observed in the 1 H NMR spectrum of 1 ( Figure 6).
In detail, immediately after the mixing of 1 and 2 + we observed the sharpening of all signals and the appearance of an AX system at 5.50/6.70 ppm attributable to aromatic H-atoms of the axle 2 + shielded inside the calixarene cavity. These changes were indicative of the formation of a pseudorotaxane 2 + 1. With this result in hand, we turned our attention to the conformation adopted by the calix [6]arene-wheel 1 in pseudorotaxane 2 + 1. A 2D COSY spectrum of 1:1 mixture of 1 and 2 + , immediately after mixing in CDCl 3 , revealed the presence of a single AX systems at 3.53/4.73, which correlates with a carbon resonance at 28.9 ppm, respectively, due to the ArCH 2 Ar methylene groups between syn-oriented Ar rings. A close inspection of the 2D HSQC spectrum revealed the presence of a cross-peak at 3.93/36.5 ppm attributable to an ArCH 2 Ar methylene bridge between anti-oriented Ar rings. These data clearly indicate that calixarene-wheel 1 adopts the 1,2,3-alternate conformation in pseudorotaxane 2 + 1 1,2,3-alt ( Figure 5 and Figure 6).
A further inspection of the 1D and 2D (COSY-45 and HSQC) spectra of the 1:1 mixture of 1 and 2 + in CDCl 3 immediately after mixing, revealed the presence of a less abundant pseudo [2]rotaxane species in which probably the calixarene wheel 1 adopts a cone conformation 2 + 1 cone ( Figure 5). Initially, the ratio between the two isomeric pseudorotaxane 2 + 1 cone /2 + 1 1,2,3-alt is 1/20, as calculated by integration of the corresponding 1 H NMR signals. Interestingly, after 10 h at 298 K (Figure 6), the intensity of the 1 H NMR signals of pseudorotaxane 2 + 1 1,2,3-alt was decreased while that of 2 + 1 cone was increased. After 18 h at 298 K, the disappearance of 2 + 1 1,2,3-alt was complete and only 2 + 1 cone pseudorotaxane could be detected by 1D and 2D NMR studies ( Figure 6). In fact, a 2D COSY spectrum of the 1:1 mixture of 1 and 2 + in CDCl 3 , after 18 h at 298 K, showed the presence of an ArCH 2 Ar AX system at 3.47/4.62 ppm which correlates in the HSQC spectrum with a carbon resonance at 28.4 ppm related to syn-oriented Ar rings. An AX system was present in the COSY spectrum at 4.78/5.68 ppm attributable to aromatic protons of the axle 2 + shielded inside the calixarene cavity. This shielded AX system correlates in the HSQC spectrum with aromatic carbon resonances at 129.8 and 126.8 ppm, respectively.
The 1 H NMR spectrum of the mixture of 1 and 2 + in CDCl 3 remained unchanged after 48 h at 298 K, thus showing that the system had reached the equilibrium condition. At this point, an apparent association constant of 6.2 ± 0.3 × 10 3 M -1 was calculated by quantitative 1 H NMR analysis (tetrachloroethane as internal standard) [37] for the formation of 2 + 1 cone pseudorotaxane. In conclusion, after the initial formation of the kinetically favored pseudorotaxane 2 + 1 1,2,3-alt (Figure 5), the thermodynamic pseudorotaxane 2 + 1 cone prevails ( Figure 5 and Figure 6). As demonstrated above, the 1,2,3-alternate conformation of 1 is the most populated in solution, consequently, the threading of this conformation, besides being faster, it is also favored by its abundance in solution.

+ 1 1,2,3-alt
and a subsequent re-threading with 1 in a cone conformation; b) a direct conformational interconversion between the 1,2,3-alternate and cone conformations of the calixarene wheel 1 in both 2 + 1 pseudorotaxanes. Previously reported data [34] clearly showed that the mechanism "b" in Figure 5 can be ruled out because the presence of an axle inside the cavity of 1 impedes the "through-the-annulus" passage of both rims of 1. From this consideration, we concluded that the two pseudorotaxanes 2 + 1 1,2,3-alt and 2 + 1 cone can be considered as two atropoisomeric forms. In fact, the interconversion between them cannot be obtained by simple rotation around chemical bonds of the calixarene wheel, which is blocked by the presence of the axle inside its cavity.
As evidenced for axle 2 + , also in this case, after the initial formation of the kinetic pseudorotaxane 3 + 1 1,2,3-alt (Figure 10), the thermodynamic atropoisomer 3 + 1 cone prevails. However, differently from the 2 + case where the kinetic product was no longer detectable in the final equilibrium mixture, here a size- able amount of the kinetic pseudorotaxane 3 + 1 1,2,3-alt can be observed at the equilibrium indicating a smaller energy difference with respect to the thermodynamic atropoisomer 3 + 1 cone . This can be ascribed to a higher destabilization of the cone atropoisomer due to a higher number of unfavourable "fluorophobic" interactions between the CF 3 group and the t-Bu-Ar moieties.

Conclusion
We have here reported a study on isomeric pseudorotaxanes in which the isomerism arises by the different conformation adopted by the calix [6]arene wheel. Among the eight possible discrete conformations of the calix [6]arene macrocycle, the cone and 1,2,3-alternate ones were observed in the pseudorotaxane architectures obtained by threading a hexahexyloxycalix [6]arene with axles bearing biphenyl or trifluoromethylbenzyl moieties. The interconversion between the cone and 1,2,3-alternate conformations occurs, in free calix [6]arene, by means of the "oxygen-through-the-annulus" and/or "p-substituent-through-the-annulus" passages. The presence of the ammonium axles inside the calixarene cavity prevents these passages; consequently two atropoisomeric pseudorotaxanes were formed. We showed that the interconversion between the two atropoisomeric pseudorotaxanes can only occur through a mechanism of de-threading/re-threading of the axle. In all the examined cases, the 1,2,3-alternate and cone atropoisomers are the kinetic and thermodynamic pseudorotaxane, respectively. We do believe that novel and intriguing calixarene-based mechanomolecules, with expanded properties or functions, could be obtained by an appropriate stoppering or catenation of such atropoisomeric pseudorotaxanes. General procedure for the preparation of 2 + and 3 + ·TFPB − salts Derivative 4 (or 5, 2.2 mmol) was dissolved at 60 °C in liquid (Me 3 Si) 2 NH (0.71 g, 4.4 mmol, 0.92 mL), LiClO 4 (0.02 g, 2.2 mmol) was added and the reaction was kept under stirring at 60 °C until a white solid was formed (30 min). The solution was allowed to cool down at room temperature and dry MeOH (4.0 mL) was added. The mixture was kept under stirring for 2 h and then cooled at 0 °C. NaBH 4 (1.12 g, 11.0 mmol) was added and the mixture was kept under stirring at 0 °C for 15 min and then allowed to warm up at room temperature. After 2 hours the solvent was removed, the solid was dissolved in ethyl acetate (100 mL) and washed with an aqueous saturated solution of NaHCO 3 (100 mL) and H 2 O (50 mL). The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure, to give secondary amine derivative. Amine was used without further purification in the next step. Secondary amine derivative (1.16 mmol) was dissolved in MeOH (20 mL) at room temperature and an aqueous solution of HCl (37% w/w, 0.20 mL) was added dropwise. The mixture was kept under stirring for 30 min, until the formation of a white precipitate. The solid was collected by filtration, washed with MeOH (10 mL) and CH 3 CN (10 mL) and dried under vacuum to give the ammonium chloride derivative. The chloride salt (0.68 mmol) and sodium tetrakis [3,5- General procedure for the preparation of pseudorotaxane derivatives The calixarene derivative 1 (3.0 mM) and ammonium salt 2 + or 3 + (3.0 mM) were dissolved in CDCl 3 (0.5 mL). Each solution was sonicated for 15 min at room temperature and then was transferred into a NMR tube for 1D and 2D NMR spectra acquisition.
Determination of apparent K ass value for pseudorotaxanes 2 + 1 cone , 3 + 1 cone and 3 + 1 1,2,3-alt , by quantitative 1 H NMR analysis. The sample was prepared by dissolving calixarene 1 (3.0 × 10 −3 M) and the ammonium TFPB salt 2 + or 3 + (3.0 × 10 −3 M) in CDCl 3 (0.5 mL) containing 1.0 μL of TCHE (d = 1.596 g/mL) as an internal standard. The complex concen-tration [complex] was evaluated by integration of the 1 H NMR signal of TCHE versus the signals of the pseudorotaxane. The following equation was used to obtain the moles of the complex: where G a = grams of TCHE, G b = grams of pseudorotaxane, F a and F b = areas of the signal of the TCHE and shielded aromatic protons of axle inside the calixarene cavity, N a and N b = numbers of nuclei that cause the signals (N a for TCHE; N b for pseudorotaxane) and M a and M b = molecular masses of TCHE (a) and pseudorotaxane (b).

Supporting Information
Supporting Information File 1 VT NMR studies of hexyloxycalix [6]arene 1, 2D COSY and HSQC spectra of atropoisomeric pseudorotaxanes, details of DFT calculations and atomic coordinates.