Molecular Russian dolls

The host-guest recognition between two macrocycles to form hierarchical non-intertwined ring-in-ring assemblies remains an interesting and challenging target in noncovalent synthesis. Herein, we report the design and characterization of a box-in-box assembly on the basis of host-guest radical-pairing interactions between two rigid diradical dicationic cyclophanes. One striking feature of the box-in-box complex is its ability to host various 1,4-disubstituted benzene derivatives inside as a third component in the cavity of the smaller of the two diradical dicationic cyclophanes to produce hierarchical Russian doll like assemblies. These results highlight the utility of matching the dimensions of two different cyclophanes as an efficient approach for developing new hybrid supramolecular assemblies with radical-paired ring-in-ring complexes and smaller neutral guest molecules.

Herein, we report the design and synthesis of a rigid tetracationic cyclophane 1 4+ with a centroid-to-centroid distance ( Fig. 1) between two BIPY 2+ units of 13.1 Å, which, in its diradical dicationic state 1 2(+•) , is ideal for encapsulating CBPQT 2 (+•) . The strong radical-pairing interactions between 1 2(+•) and CBPQT 2(+•) has led to the formation of a unique tetraradical tetracationic, box-in-box complex. Interestingly, this box-in-box complex can accommodate small aromatic guest molecules inside the void of the CBPQT 2(+•) component to generate a series of tetraradical tetracationic Russian doll assemblies, both in solution and in the solid state. This kind of complexation is rare in the case of organic compounds [26][27][28][29][30][31] .
Solution phase characterization of the box-in-box complex. The association between 1 2(+•) and CBPQT 2(+•) was investigated in the first instance by UV-Vis-NIR spectroscopy. Solutions of 1·4PF 6 (0.5 mM) and CBPQT·4PF 6 (0.5 mM) in MeCN were reduced by Zn dust to generate the corresponding radical and CBPQT 2(+•) . In order to estimate the binding affinity between 1 2(+•) and CBPQT 2(+•) , UV-Vis-NIR absorption titrations were carried out by monitoring the change of the NIR absorption band centered on 910 nm. The resulting data could be made to fit a 1:1 isotherm to give ( Fig. 3b) 44,49 to be diamagnetic, based on EPR measurements, the tetraradical tetracationic host-guest complex [CBPQT ⊂ 1] 4(+•) was also expected to be diamagnetic. Thus, we sought to characterize the host-guest complex by 1 H NMR spectroscopy, as we have previously succeeded 45 in doing for the tetraradical tetracationic state of a rotaxane, based on the [m-CBPQT ⊂ SqBox] 4(+•) recognition motif. At room temperature, however, only a single broad peak near 7.7 ppm was observed in the spectrum (Fig. 4) of a 1:1 mixture of 1 2(+•) and CBPQT 2(+•) in CD 3 CN, presumably because of a small thermal population of a paramagnetic state at room temperature 45,49 . Upon cooling to -20°C, the signals of the complex are individually resolved, yet remain broad until further cooling to -40°C, at which point all of the resonances of the host-guest complex are displayed (Fig. 4) as sharp signals.
is the first radical-pairing-based host-guest complex to be characterized by 1 H NMR spectroscopy, we sought to probe the limits of this technique more deeply for studying supramolecular assemblies featuring radical-radical interactions. Thus, 1 H-1 H COSY NMR spectra were recorded (Supplementary Figure 6) at -40°C in order to assign the peaks of the host-guest complex. Noticeably, both signals for the aromatic and CH 2 protons on CBPQT 2(+•) are shifted significantly to higher field (Fig. 4) as a consequence of the stronger shielding effect of the host-guest interactions compared with that of 1 2(+•) . Resonances for the meta positions of the pyridinium units in 1 2(+•) and CBPQT 2(+•) which were located at 5.22 (peak b in Fig. 4) and 3.28 ppm (peak f in Fig. 4), respectively, are shifted to considerably higher fields than those of the ortho protons of the pyridinium units (7.73 ppm for peak a and 7.34 ppm for peak e in Fig. 4). This observation is in line with the solid-state superstructure (Fig. 6, see below) of the complex [CBPQT ⊂ 1] 4(+•) , which shows that the BIPY +• units in the radical dimers (BIPY +• ) 2 are nearly perpendicular to each other, torsion angle 84°, such that the meta CHs of the BIPY +• units in 1 2(+•) and CBPQT 2(+•) are strongly shielded 45 , while the ortho CHs are not shielded.
CW-EPR spectroscopy experiments were carried out in order to probe the electronic structure of [CBPQT ⊂ 1] 4(+•) . The EPR spectrum of CBPQT 2(+•) (298 K, 0.50 mM, Supplementary Figure 22) displays almost no hyperfine structure. This observation is a characteristic result of the unpaired electrons in the two BIPY +• units being close enough together for electronic overlap such that spin-exchange interactions between them dominate the spectrum 39 . The EPR (298 K, 0.50 mM, Supplementary Figure 22) spectrum of 1 2(+•) exhibits, however, hyperfine splitting which resembles that of other N,N′-dialkyl substituted BIPY +• radical cations reported in the literature 39,46 , since the relatively longer separation distance between two BIPY +• units leads to the absence of efficient overlap for spin-exchange interactions. In comparison, the EPR spectrum (298 K, 0.25 mM each, Supplementary Figure 22) of a 1:1 mixture of 1 2(+•) and CBPQT 2(+•) exhibits relatively minimal hyperfine structure. This implies a disruption of the spin-exchange interactions between the two BIPY +• radicals by an interaction (complexation) between 1 2(+•) and CBPQT 2(+•) . Variable-temperature EPR spectra (Fig. 5a) of the 1:1 mixture of 1 2(+•) and CBPQT 2(+•) in MeCN was then measured at temperatures ranging from -20 to 80°C. The EPR spectra at −20°C of the 1:1 mixture displays almost no microwave absorption by unpaired radicals. This indicates that there is strong association of the cyclophanes to form the radicalpaired ground state of the complex [CBPQT ⊂ 1] 4(+•) , an observation which is consistent with the VT-NMR results. Upon heating from −20 to 80°C, the EPR signal of the mixture gradually increased in intensity, along with an increase in the appearance of hyperfine structure. This observation is ascribed to the temperature-induced dissociation of the box-in-box complex into its individual radical cyclophane components.
In order to gain insight into the redox processes involved in the assembly and disassembly of [CBPQT ⊂ 1] 4(+•) , cyclic voltammetry (CV) experiments were performed using ferrocene as the internal redox standard. Both CBPQT·4PF 6 and 1·4PF 6 (concentration: 0.20 mM in MeCN containing 0.1 M NBu 4 PF 6 , scan rate: 0.2 V/s) exhibit (Fig. 5b, c) similar reversible redox waves, though the waves for CBPQT·4PF 6 are more positively shifted than those of 1·4PF 6 , presumably because of its higher ring strain, resulting from the smaller ring size. CVs of an equimolar mixture of CBPQT·4PF 6 and 1·4PF 6 were also measured (Fig. 5d) at a concentration of 0.20 mM for each box. With a scan rate of 0.2 V/ s, the equimolar mixture displays two sharp reversible redox waves, similar to those of the individual boxes, but shifted to more positive potentials. The shifts in the potentials of the redox waves for the mixture of CBPQT·4PF 6 and 1·4PF 6 can be rationalized by the formation of a tetraradical tetracationic host-guest complex since radical-pairing interactions will lend stability to the radical cationic redox states, as observed previously 39,46 for other radical-based host-guest complexes. Furthermore, CV experiments which were carried out at different scan rates show scan rate-dependent CV behavior Single-crystal X-ray diffraction analysis revealed (Fig. 6, Supplementary Figure 16) a solid-state superstructure in which CBPQT 2(+•) is encapsulated inside the cavity of the 1 2(+•) ring to form a 1:1 host-guest inclusion complex that is consistent with the results found in solution.
Formation of Russian doll assembly. Notably, the solid-state superstructure of the box-in-box complex [CBPQT ⊂ 1] 4(+•) , confirmed the presence of a void inside of the smaller CBPQT 2 (+•) cyclophane component that is large enough to potentially accommodate a second guest. We thus sought out guest molecules that could occupy this cavity to form a Russian doll-like assembly. Radical cationic dimethyl viologen (MV +• ) was firstly employed as the guest in order to investigate the possibility of forming a pentaradical pentacationic [MV ⊂ CBPQT ⊂ 1] 5(+•) complex. Neither UV-Vis-NIR spectroscopy nor X-ray crystallography, however, indicated the formation of this threecomponent assembly. We attribute this observation to the limited space between the p-phenylene and bis(p-phenylene)ethyne spacers of the two cyclophanes in [CBPQT ⊂ 1] 4(+•) , which might not provide a large enough window for MV +• to thread.
Smaller neutral guest molecules, such as 1,4-dichlorobenzene, were employed in an effort to find guests that could fit into the cavity of the [CBPQT ⊂ 1] 4(+•) complex. These investigations were facilitated by the ability to observe well-defined 1 H NMR spectra of [CBPQT ⊂ 1] 4(+•) at reduced temperatures, and thus, 1 equiv 1,4-dichlorobenzene was added to a 1.0 mM solution of [CBPQT ⊂ 1] 4(+•) in CD 3 CN, and VT 1 H NMR spectra were recorded. Upon decreasing the temperature from 25 to −40°C, the signals (Fig. 7) of a three-component [p-C 6 H 4 Cl 2 ⊂ CBPQT ⊂ 1] 4(+•) complex gradually appeared and sharpened into well-resolved signals. The formation of the three-component complex was evident from the change in the chemical shifts of all the peaks of the two cyclophane components from those that were observed (Fig. 4) for the two component complex [CBPQT ⊂ 1] 4(+•) in the absence of 1,4-dichlorobenzene. The signals of the BIPY +• units, in particular, were shifted significantly to higher fields. The encapsulating of 1,4-dichlorobenzene was further confirmed by the observation (Fig. 7)  One matter that is worth investigating is whether the inclusion of the third component guest (p-C 6 H 4 Cl 2 ) will affect the binding affinity between CBPQT 2(+•) and 1 2(+•) . Thus, we repeated the UV-Vis-NIR titration experiments with p-C 6 H 4 Cl 2 presented in the solution. By titrating CBPQT 2(+•) into the solution mixture of 1 2(+•) and a number of equivalents of p-C 6 H 4 Cl 2 , similar 1:1 fitting isotherms can be applied to the titration data, and "apparent" binding constants were obtained. Notably, the existence of p-C 6 H 4 Cl 2 guests did, indeed, increase the binding affinity between 1 2(+•) and CBPQT 2(+•) ; the more equivalents of p-C 6 H 4 Cl 2 that were added, the more the binding affinity increased. For instance, when 2.0 eq. p-C 6 H 4 Cl 2 was present, the "apparent" binding constant was found to be (3.8 ± 0.3) × 10 4 M -1 (Supplementary Figure 11), which is 1.3 times higher than that of the box-in-box complex without p-C 6 H 4 Cl 2 ; and when the equivalents of p-C 6 H 4 Cl 2 were 6.7 and 20, the "apparent" binding constant increased to (6.2 ± 0.7) × 10 4 M -1 (Supplementary Figure 12) and (9.0 ± 1.2) × 10 4 M -1 (Supplementary Figure 13), respectively. These results demonstrate that the inclusion of p- 1] 4(+•) complex to form the desired Russian doll-like [p-C 6 H 4 Cl 2 ⊂ CBPQT ⊂ 1] 4(+•) complex. The co-conformation of the two cyclophane components within the three-component complex remains almost the same (Fig. 6) as in the complex [CBPQT ⊂ 1] 4(+•) in the absence of an additional guest, with the three-component complex exhibiting a contact distance between BIPY •+ units of 3.05 Å and a torsion angle of 86°. The distance between 1,4-dichlorobenzene and the adjacent BIPY •+ units is 3.55 Å, an observation that indicates the presence of [π···π] interactions between 1,4-dichlorobenzene and CBPQT 2(+•) . The two Cl atoms of the guest are located at the "windows" between the two cyclophanes and exhibit short Cl···H contact distances (2.97 and 3.03 Å) with the two ortho H-atoms of the bis (phenylene)ethyne units. This observation indicates the existence of weak [C-H···Cl] hydrogen bonds between the diphenylethyne units and dichlorobenzene guest, which providing additional stabilization of the Russian doll assembly. The successful formation of a three-component complex with 1,4-dichlorobenzene prompted us to explore other 1,4-substituted benzene derivatives-namely, 1,4-dibromobenzene, dimethyl terephthalate, 1,4-bis(allyloxy)benzene and bis(propargyl)-terephthalateas guest molecules to bind inside the [CBPQT ⊂ 1] 4(+•) complex. All these molecules were found to form Russian doll assemblies (Fig. 8, Supplementary Figure 17 -21) in the solid-state that are similar to the three-component complex formed with 1,4dichlorobenzene. The contact distances between the BIPY +• units of two cyclophane components are nearly identical (3.04-3.05 Å, Fig. 8) for all of the Russian doll assemblies. The torsion angles, however, vary significantly for different guest molecules-86°for 1,4-dichlorobenzene, 78°for 1,4-dibromobenzene, 74°for 1,4-bis(allyloxy)benzene, 70°for 1,4-bis(propargyl)-terephthalate and 66°for dimethyl terephthalate-reflecting the steric influence of the 1,4-substituents on these benzene derivatives. In addition, the accommodation of different guest molecules significantly influences the packing properties (Supplementary Figure 17-21) of the complexes. It is noteworthy that [1,4-bis(allyloxy)benzene ⊂ CBPQT ⊂ 1] 4(+•) and [1,4-bis(propargyl)-terephthalate ⊂ CBPQT ⊂ 1] 4(+•) are ring-in-ring-type pseudo [3]rotaxanes with vinyl or alkynyl functional groups at the end of the dumbbells, making these assemblies potential precursors for preparing new mechanically interlocked molecules 45,50 and redox-controlled molecular machines 51 .

Discussion
As detailed above, we have synthesized a tetracationic cyclophane that was designed to act as a size-complementary host for recognizing the smaller CBPQT 4+ cyclophane upon reduction of both cyclophanes to their diradical dicationic states. Radicalpairing interactions between the two cyclophanes drives the formation of a novel tetraradical tetracationic box-in-box complex [CBPQT ⊂ 1] 4(+•) , which was investigated thoroughly in solution by spectroscopic techniques (UV-Vis-NIR, VT-EPR, VT-1 H-NMR) and cyclic voltammetry. An association constant of K a = (1.6 ± 0.2) × 10 4 M -1 was determined on the basis of UV-Vis-NIR titration experiments, and EPR measurements confirmed strong radical-pairing between the cyclophanes to produce a diamagnetic assembly. Remarkably, the radical-pairing interactions are strong enough to permit characterization of [CBPQT ⊂ 1] 4(+•) by 1 H NMR spectroscopy-the first time this technique has been used for characterizing a supramolecular assembly held together by radical-radical interactions. Characterization by 1 H NMR spectroscopy provided detailed information about the co-conformation of [CBPQT ⊂ 1] 4(+•) in solution, which was consistent with the solid-state superstructure that was determined by single-crystal XRD analysis.
The most notable feature of the box-in-box complex is its ability to bind various 1,4-disubstituted benzene derivatives inside the cavity of the smaller cyclophane to form hierarchical Russian doll assemblies, which is quite rare in supramolecular systems. Besides, the inclusion of third component guests within the cavity of the box-in-box complex can help to stabilize the assembled structures. These three-component assemblies were characterized in the solid-state by single-crystal XRD analysis, as well as in solution by 1 H NMR spectroscopy. The formation of these hierarchical complexes highlights the box-in-box complex as an attractive motif for forming sophisticated supramolecular assemblies that could be used for designing complicated new mechanically interlocked structures and redox-controllable molecular machines. Thus, this research demonstrates the utility of precisely designing the dimensions of a host for binding a specific guest, such as a smaller cyclophane, that imbues the resulting host-guest complex with appealing properties and functionalities.
Synthesis of 3•2PF 6 . 4,4′-Bipyridine (4.0 g, 25.6 mmol) was dissolved in MeCN (60 mL), and the solution was heated to 90°C. Compound 2 (1.3 g, 4.12 mmol) was added in three portions within 1 h. The solution was cooled to room temperature after refluxing for 24 h, and then TBACl (2.5 g, 9.0 mmol) was added. UV-Vis-NIR titration. Stock solutions of the fully oxidized viologen derivatives CBPQT•4PF 6 , 1•4PF 6 were prepared in an N 2 glovebox. The stock solutions were reduced over activated Zn dust for 10-15 min with stirring and then filtered to provide deep blue solutions of CBPQT 2(+•) or 1 2(+•) . Syringes were employed to measure and dilute the radical stock solutions to the desired concentrations prior to measurements. Spectra were recorded from 1300 to 400 nm in a sealed 2 mm path length cells during titrating CBPQT 2(+•) (0 to 10 equiv) into 1 2(+•) . Binding constant was obtained by fitting fit a 1:1 isotherm according to literature 47 .
EPR measurement. Solutions of CBPQT 2(+•) and 1 2(+•) were prepared in N 2 glovebox in the same way as was described for preparing the samples utilized for UV-Vis-NIR measurements. After mixing and/or diluting the radical samples to the desired concentration, 100 μL of each sample was transferred to a quartz EPR tube by syringe. The tubes were sealed with UV-cure resin under an N 2 atmosphere.
Cyclic voltammetry measurement. Samples for cyclic voltammetry were prepared using an electrolyte solution of 0.1 M Bu 4 NPF 6 in MeCN that was sparged with Ar to remove O 2 . The cyclic voltammograms presented in the main text of the manuscript were recorded under Ar or N 2 using a glassy carbon working electrode, a Pt wire or Pt mesh counter electrode, a silver wire quasi-reference electrode, and an internal standard of ferrocene.
VT-NMR measurement. The sample solutions were prepared in N 2 glovebox with a similar procedure as described for preparing the samples utilized for UV-Vis-NIR measurements. Variable-temperature 1 H NMR spectra and low temperature 1 H-1 H COSY NMR were recorded using an Agilent DD2 spectrometer with a 600 MHz working frequency for 1 H nuclei.
Crystallizations and X-ray analyses for all complexes. For 1•4PF 6 : Single crystals were grown on the bench-top by slow vapor diffusion of i Pr 2 O into a 1.0 mM solution of 1•4PF 6 in MeCN over the course of a week. For [CBPQT ⊂ 1]•4PF 6 and all the five Russian doll assemblies: Excess of activated Zn dust was added to a mixture of CBPQT•4PF 6 (1.1 mg, 1.0 µmol), 1•4PF 6 (1.3 mg, 1.0 µmol) together without additional guests ([CBPQT ⊂ 1]•4PF 6 ) or with 4 equiv of the corresponding 1,4-disubstituted benzene derivatives in an N 2 glovebox, and the mixtures were stirred for 20 min. After filtering, the purple solutions were kept under an atmosphere of i Pr 2 O at room temperature for a week to allow slow vapor diffusion to occur. The crystals, which appeared in the tubes, were selected and mounted using oil (Infineum V8512) on a glass fiber and transferred to the cold gas stream cooled by liquid N 2 on Bruker APEX-II CCDs with MX optics Mo-K α or Cu-K α radiation. The structures were solved by direct methods and refined subsequently using OLEX2 software. CCDC 1851540-185146 contain the supplementary crystallographic data for this article. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Data availability
All the data generated or analyzed during this study are included in this published article (and its supplementary information files) or available from the authors upon reasonable request. The crystallographic data in this study have been deposited in the Cambridge Structural Database under entry IDs CCDC 1851540-185146.