Macrocyclic shape-persistency of cyclo[6]aramide results in enhanced multipoint recognition for the highly efficient template-directed synthesis of rotaxanes

The importance of macrocyclic shape-persistency in novel host–guest systems for the highly efficient template-directed synthesis of rotaxanes has been revealed.


Introduction
The development of mechanically interlocked molecules (MIMs) has invariably been accompanied by emerging recognition motifs and template-directed synthetic protocols. 1 Among various factors, the combination of a macrocycle and a thread component with sufficient binding affinity constitutes a crucial determinant to create such motifs. In this regard, many elegant examples have been reported. These include the use of macrocyclic hosts such as crown ethers, 2 tetracationic cyclophanes, 3 calixarenes, 4 cucurbiturils, 5 cyclodextrins, 6 and pillarenes 7 to interact with the chosen thread components. However, uncovering simple and efficient recognition motifs still represents a grand challenge for the synthesis of MIMs, especially those involving non-metal coordination for extremely compact interlocked molecules, with high atom economy.
Thus far, most known examples of highly efficient, templatedirected synthesis of [3]rotaxanes have relied on metal-ligand coordination. 8 In all these cases, the "wheels" employed are based on macrocyclic molecules that are nonplanar and fairly exible conformationally. In recent years, shape-persistent macrocycles, with noncollapsible and geometrically well-dened skeletons, such as phenylacetylene macrocycles, 9 have attracted considerable attention due to their intriguing functions, including supramolecular gelation, 10 channelized transportation, 11 organic catalysis, 12 molecular recognition 13 and multifunctional selfassembly. 14 Many shape-persistent macrocycles have well-dened surface topography and nanosized cavities with preorganized binding sites, and thus may exhibit enhanced complexation. 15 Inspired by the pioneering work on synthesizing MIMs using exible aromatic amide macrocycles 16 or H-bonded oligoamide foldamers, 17 we reasoned that a shape-persistent macrocycle, when serving as a wheel component for a MIM, may overcome the limit of known systems by engaging effectively in cooperative recognition interactions. However, introducing two-dimensional (2D) shape-persistent macrocycles into interlocked structures still presents a signicant challenge. To the best of our knowledge, few examples of MIMs based on these macrocycles are known, 18 especially [3]rotaxanes. In fact, there is only one report, based on a pentagonal cyanostar macrocycle, on forming [3] rotaxanes. 18a H-Bonded aromatic amide macrocycles 19 are a large class of recently emerged 2D shape-persistent cyclic compounds featuring full amide linkages with rigid backbones enforced through intramolecular hydrogen bonds. Among them, cyclo [6] aramides, the smallest member of the aromatic oligoamide macrocycles 20 with the amide carbonyl oxygens pointing inwards, are particularly noteworthy. They resemble a crown ether in their oxygen-rich cavities, but differ considerably in their conformational rigidity. In particular, the highly favorable intramolecular H-bond assisted macrocyclization strategy makes numerous geometrically well-dened macrocycles readily available, which facilitates their further applications. With a well-dened cavity of ca. 8Å in diameter, large p-surfaces and precisely positioned binding sites, these macrocycles have displayed rich host-guest (H-G) chemistry. Our recent studies revealed that cyclo[6]aramides could bind organic cations and hydrogen bond donors. 21 Especially notable is the complex consisting of such macrocycles and diquat, an isomer of paraquat, with 2 : 1 stoichiometry, in which a diquat molecule resides between two macrocyclic molecules rather than threads into their cavities due to electronic repulsion and steric hindrance. 22 Paraquat is one of the most widely used guests for studying H-G interactions. 23 Despite numerous reports on paraquat recognition, threading two rings on a single paraquat molecule with high binding affinity both in solution and in the solid state is still very difficult to achieve. The cryptand reported by Gibson and co-workers could form a 2 : 1 H-G complex only observed in the crystal structure and the highest binding constant for 1 : 1 stoichiometry achieved so far is 5.0 Â 10 6 M À1 in acetone. 24 A crown ether, bis-p-xylyl[26]crown-6, was used by Chiu and co-workers to form a [3]pseudorotaxanelike complex both in solution and in the solid state. However, only a very low binding constant (K 1 ¼ 700 M À1 and K 2 ¼ 60 M À1 ) was observed in acetonitrile. 25 So far no successful examples have been reported on creating a tight 2 : 1 binding motif for constructing constrained [3]rotaxanes based on a single paraquat unit. Herein we report that cyclo[6]aramides, with their persistent shape and geometrically well-dened electron-rich cavity (Scheme 1a), could act as powerful hosts for the tight binding of bipyridinium salts in a 2 : 1 binding mode both in the solid state and in solution with exceedingly high binding constants. More importantly, this unique recognition motif has led to the template-directed synthesis of compact [3]rotaxanes in excellent yields based on either a "click-capping" or "facile onepot" approach (Scheme 1b).

Results and discussion
Evidence for 2 : 1 cooperative host-guest complexation The rst sign indicating the H-G interaction came from a color change upon adding paraquat G1 to a solution of cyclo[6]aramide 1 in acetone. An abrupt change from clear to light yellow was observed, indicating that a charge transfer (CT) interaction happened between 1 and G1. A CT band in the UV-vis absorption spectrum conrms the formation of the H-G inclusion complex (Fig. S69 †). Then, the formation of the H-G complex was further explored using 1 H NMR spectroscopy (Fig. 1). When 1.0 equiv. of 1 was added to a 2.0 mM solution of G1, two sets of signals from the bipyridinium ion were observed, which evolved into one set of signals with 2 equiv. of 1, indicating the slow exchange of the complex on the NMR time scale. Commensurate with this change is the appreciable shi for the aromatic and amide protons of the host upon guest binding. The binding event was supported with 2D nuclear overhauser effect spectroscopy (NOESY), which revealed through-space NOEs between bipyridinium protons and the internal aromatic protons H a and H b of G1-G4 ( Fig. S55-S64 †). Such through-space NOE contacts can transpire only if these two macrocycles are mutually parallel but orthogonal to G1. Two-dimensional diffusion ordered spectroscopic (2D-DOSY) analysis provided additional evidence for the formation of very stable complexes between 1 and G1-G4 . For example, in the case of the complex 1 2 I G1, the maximum change in absorbency was observed at 0.67, indicating a macrocycle-cation ratio of 2 : 1. Examining the 2 : 1 mixture of 1 and G1 using matrix-assisted laser ionization time of ight mass spectrometry (MALDI-TOF-MS) uncovered the peak with the highest intensity at m/z ¼ 5005.219, corresponding to the complex [1 2 + G1 À PF 6 ] + . The above results, taken in concert, clearly demonstrate the formation of a 2 : 1 complex 1 2 I G1. The same 2 : 1 stoichiometry was observed with guests G2 and G4, but G3 only shows 1 : 1 stoichiometry with 1 ( Fig. S103-S109 †). Another interesting observation made on complex 1 2 I G1 is its reversible redox-responsiveness, which was realized by the addition and removal of Zn powder (Fig. S143 †).
The binding constants K 1 and K 2 for the complexation of cyclo[6]aramide 1 with bipyridinium salts G1-G4 were obtained using UV-vis titration methods (Table 1). The UV-vis titration experiments revealed surprisingly high binding constants, K 1 ¼ 3.49 Â 10 7 M À1 and K 2 ¼ 1.09 Â 10 6 M À1 , for complex 1 2 I G1. The binding of G1 with the second macrocycle is accompanied by a slightly negative cooperative effect (4K 2 /K 1 < 1), 26 which is probably caused by the remaining pyridinium unit that has become less electron decient aer the threading of the rst macrocycle. To provide insight into the role of the second pyridinium moiety when the rst site is occupied, G2, with one positive charge, was examined for binding to 1. Results from UV-vis titration show that, as compared to those of 1 2 I G1, the binding constants for G2 and 1 are drastically reduced to K 1 ¼ 3.62 Â 10 4 M À1 and K 2 ¼ 2.28 Â 10 4 M À1 , which indicates the pivotal role played by the second positive charge of G1 for the high stability of complex 1 2 I G1. In addition, the positive cooperativity (4K 2 /K 1 ¼ 2.51) observed for 1 2 I G2, along with the similar K a values of complex 1 I G3 to that of K 1 for 1 2 I G1, suggests that additional inter-macrocycle p-p stacking may also assist the binding of the second macrocycle in the formation of 1 2 I G2. It is worth noting that G4 gives the highest binding constants for binding 1 (K 1 ¼ 1.68 Â 10 8 M À1 and K 2 ¼ 2.47 Â 10 7 M À1 ), which should greatly facilitate the synthesis of rotaxanes (Fig. S83-S95 and Table S1 †). A similar trend in binding constants was observed in a competitive solvent (acetone-d 6 /DMSO-d 6 , 9/1, v/v) ( Table 1 Cooperativity a (4K 2 /K 1 ) c 1 2 I G1 Acetone 2 : 1 3.49 Â 10 7 1.09 Â 10 6 3.80 Â 10 13 0.12 1 2 I G1 Acetone/DMSO (9/1, v/v) 2 : 1 3.29 Â 10 6 3.72 Â 10 5 1.22 Â 10 12 0.45 1 2 I G2 prepared from pyridinium salts and macrocycle 1 show that the C]O stretching frequency shi induced by complex formation is in the order of G4 > G1 > G2 > G3 (Fig. S110-S113 and Table  S1 †), which agrees well with the difference in the binding affinities of these guests with macrocycle 1. In order to demonstrate the crucial role played by shape persistency, cyclo [6]aramide 5, which is partially H-bonded and bears two rotatable amide groups, was synthesized. Since the depletion of partial intramolecular H-bonds results in free rotation of the two amide groups, the shape-persistency as observed in 1 should be attenuated to a signicant extent. Indeed, the results from the binding experiments show that it binds G4 in a 2 : 1 binding mode (Fig. S81-S82 †) with binding constants of K 1 ¼ 7.29 Â 10 4 M À1 and K 2 ¼ 2.50 Â 10 3 M À1 (Table 1 and Fig. S95 and S96 †), which are four orders of magnitude lower than that of complex 1 2 I G4. This signicantly reduced binding affinity as compared to 1 strongly suggests the importance of shape-persistency in the binding event. Despite the great advances made in the past decades, few recognition modules have shown such unusually strong binding in organic media.

X-ray crystal structure of [3]pseudorotaxane 3 2 I G1
The slow evaporation of a solution containing 3 and G1 in a mixed solvent of acetone/CHCl 3 /methanol (10/1/0.3) afforded red crystals in 7 weeks. The analysis of the resulting solid state structure using single-crystal X-ray diffraction (XRD) experiments reveals that G1 is inserted into two macrocyclic molecules. In this complex, each of the pyridinium units is orientated orthogonally with respect to the two macrocycles and engages in multiple C-H/O H-bonding (Fig. 2a) and N + /O ion-dipole interactions that are reinforced by face to face p-p stacking interactions (3.71Å) (Fig. 2b). These hydrogen bonds all have very short donor-acceptor distances ranging from 2.30 to 2.74Å due to the rigid macrocyclic backbone constrained by intramolecular three-centre H-bonds. The length of the N + /O ion-dipole interactions varying from 3.32 to 4.73Å is indicative of strong Coulomb interaction between the amide oxygen atoms and pyridinium cations. Therefore, the strong binding affinity between cyclo[6]aramide 1 and the bipyridinium guests is attributed to the result of the cooperative interplay of multipoint non-covalent forces. The geometrically well-dened and tightly packed solid-state structure of 3 2 I G1 is also observed which is stabilized by multiple van der Waals forces between the side chains of macrocycle 3 and G1 (Fig. 2c).
"Click-capping" approach for the synthesis of rotaxanes  recognition event favours 2 : 1 stoichiometry, rather than 1 : 1, during the binding process. The highly selective formation of [3] rotaxanes with cyclo[6]aramides originates from the combination of exceedingly high binding affinities and the compact selfassembly mode. These features are rarely observed in other similar H-G systems. In addition, face to face p-p stacking interactions between the two near-planar macrocycles as observed in the crystal structure of 3 2 I G1 could provide an additional driving force that contributes to the high efficiency. It is worth noting that the preparation of [3]rotaxanes with shape-persistent macrocycles as "wheels" in such a high yield (>90%) under non-metal coordination conditions is preceded by few examples and remains a formidable task. 18a,30 "Facile one-pot" approach for the synthesis of rotaxanes The "facile one-pot" reaction, characterized by simply mixing and heating, and the absence of metallocatalysts, is another useful approach in the synthesis of rotaxanes with high yields. 31 The synthesis of [3]rotaxanes [3]R-C n (n ¼ 16 and 12) or [2] rotaxane [2]R-C 6 based on this method was achieved through mixing 2.0 equiv. of cyclo[6]aramide, 2.5 equiv. of Stopper-Br and 1.0 equiv. of 4,4 0 -bipyridine in CHCl 3 /CH 3 CN (1/1, v/v) ( Table 3, entries 1-3). Highly efficient template-directed synthesis with macrocycle 1 or 2 was achieved with an excellent yield of 85%, which is rare in the known synthesis of [3]rotaxanes. 32 Particularly noticeable was the formation of a [2]rotaxane as the only product when macrocycle 3, which bears short side chains, was used. This specicity in [2]rotaxane formation is rationalized according to the sparse dissolution of the macrocycle in the solution. Compound 3 alone was insoluble in CHCl 3 /CH 3 CN (1 : 1, v/v). However, gradual dissolution was observed to occur as the reaction progressed. This suggests a scanty concentration of macrocycle 3 in the reaction system as compared to that of macrocycle 1 or 2. Since the efficiency of forming [3]rotaxanes depends predominantly on the effective molar ratio (2 : 1) of the macrocycle and cationic guest in solution, the limited concentration of macrocyclic molecules with respect to that of the cationic axle tends to facilitate a binding process that favours a 1 : 1 binding mode, thereby leading to the specic formation of a [2]rotaxane.

X-ray crystal structure of [3]CR-C 6
Single crystals of [3]rotaxane [3]CR-C 6 were obtained by slowly diffusing methanol into an acetone solution containing [3]CR-C 6 in about 8 weeks. The X-ray structure of [3]CR-C 6 clearly shows that the thread Axle-1 penetrates through the cavities of the two neighboring macrocyclic molecules in a zigzag conformation ( Fig. 5a and b). The mechanically interlocked structure is stabilized by twenty C-H/O H-bonds and twelve N + /O ion dipole interactions (Tables S4 and S5 †). There are also a number of very weak face-to-face p-p stacking interactions (4.658Å) between the macrocyclic molecules, resulting in a well-ordered and tightly packed solid-state structure (Fig. 5c). The numerous noncovalent forces revealed from the X-ray structure work cooperatively, leading to the surprisingly high stability of the [3]rotaxanes. In fact, when a mixture of Axle-1 and macrocycle 1 was heated under reux for 3 hours in acetone-d 6 /DMSO-d 6 (9 : 1, v/v), the 1 H NMR spectra did not show any signs of the presence of rotaxanes, indicating the fact that threading does not occur (Fig. S117 †), and thus it can be inferred that [3]rotaxane [3]CR-C 6 is unlikely to experience a dethreading process under the conditions specied. The high stability is also demonstrated by the observation that complex 1 2 I G1 (or 1 2 I G4) and free macrocycle 1 were clearly seen for each of them on a TLC plate. Upon the addition of 10.0 equiv. of diethylamine (DEA) to [3]R-C 16 or [2]R-C 6 in acetone, only [2]R-C 6 caused a color change from light yellow to blue, indicating the insensitivity of [3]R-C 16 to redox responsiveness. Interestingly, triuoroacetic acid (TFA) can reverse the redox process of [2]R-C 6 (Fig. S144 †).

Computational simulation of [3]R-C 1
Since the growth of single crystals of the [3]rotaxane [3]R-C n (n ¼ 16, 12 and 6) synthesized according to the "facile one-pot" approach has proved to be extremely challenging, we resorted to molecular mechanics simulations to gain a better understanding of the noncovalent bonding interactions that direct rotaxane formation and stability. Further structural insights on [3]rotaxane [3]R-C 1 were obtained through computational simulations based on the DFT method. Our computational study indicated that [3]rotaxane [3]R-C 1 was built by assembling the two macrocyclic molecules of 4 in a near-planar conformation and the central motif Axle-1 in an interlocked orthogonal binding arrangement, which is in good agreement with the structure obtained from the single crystal of [3]CR-C 6 . Furthermore, multiple C-H/O H-bonds and N + /O ion-dipole interactions are also observed in the modelling structure which direct the [3]rotaxane formation (Fig. S153 †). It is worth noting that there are no face to face p-p stacking interactions formed between the two macrocycles, which is the same as the observation from the crystal structure of [3]CR-C 6 .

Conclusions
In conclusion, we have demonstrated the unusually high binding affinity (K a from $10 13 M À2 to $10 15 M À2 in acetone) of a novel threaded recognition motif comprising cyclo[6]aramides and bipyridinium salts in 2 : 1 (H : G) stoichiometry. The crystal structure of the [3]pseudorotaxane shows clear evidence of the high binding affinity, which is attributed as the result of the cooperative interplay of multipoint C-H/O H-bonds, N + / O ion-dipole interactions and p-p stacking interactions between the two neighboring macrocycles. Furthermore, the highly efficient synthesis of compact [3]rotaxanes achieved using either a "facile one-pot" or "click-capping" approach presents a rare example of constructing MIMs based on 2D shape-persistent macrocycles. The high efficiency of the formation of these rotaxanes highlights the unique advantage of macrocyclic shape-persistency, which results in enhanced multipoint recognition for the highly efficient synthesis of compact mechanically interlocked molecules. The concept of utilizing macrocyclic shape-persistency for boosting multipoint binding affinities for the template-directed synthesis of rotaxanes might be useful for the design of novel MIMs and the development of articial molecular machines.