Nanohoop Rotaxanes via Active Template Syntheses and Their Potential in Sensing Applications
The unique optoelectronic properties and smooth, rigid pores of macrocycles with radially oriented pi systems render them as fascinating candidates for the design of novel mechanically interlocked molecules with new properties. Herein, we report two high-yielding strategies to prepare nanohoop [2]rotaxanes, which owing to the pi-rich macrocycle, are highly emissive. Then, through a combination of x-ray crystallography, NMR, and fluorescence spectroscopy, we illustrate that metal-coordination-an intrisic property afforded by the resulting mechanical bond-can lead to molecular shuttling as well as modulate the observed fluoresence in both organic and aqueous conditions. Inspired by these findings, we design a self-immolative [2]rotaxane, which self-destructs in the presence of an analyte, eliciting a strong fluorescent turn-on response, serving as proof-of-concept for a new type of molecular sensing material. More broadly, this work highlights the conceptual advantages of combining compact π-rich macrocyclic frameworks with mechanical bonds formed via active-template syntheses.
INTRODUCTION<!>RESULTS AND DISCUSSION<!>Metal coordination with trizaole-embedded rotaxanes.<!>Conclusion<!>Supporting Information.<!>AUTHOR INFORMATION Corresponding Author
<p>Carbon-rich molecules with closed circuits of delocalized pi electrons have been of longstanding interest due to their unique optical, magnetic, and electronic properties. 1 Of this broad class of molecules, structures that have radially oriented pi systems-pi systems pointing inwards to the cavity of a macrocycle-have recently emerged as a new class of strained, nonplanar aromatic molecules with unusual properties. Specifically, our laboratory and others have synthesized molecules referred to as "carbon nanohoops" due to their structural relationship to carbon nanotubes (Figure 1a). 2 These shape persistent macrocycles can be prepared with varying size and atomic composition (Figure 1b), 3 which has unveiled their numerous size-dependent optoelectronic behavior and promising materials applications. 2h, 3n In particular, due to the tunable and bright fluorescence, 2b biocompatibility, 3i and metal binding capabilities, 3c, g, f we have a growing interest in the development of these pi-rich macrocycles for biological applications.</p><p>In the field of supramolecular chemistry, mechanically interlocked molecules (MIMs) such as catenanes, rotaxanes, and molecular knots have captured the imagination of chemists highlighted by the recent Nobel Prize in 2016. 4 A particularly intriguing consequence of threading and/or weaving molecular components is that the resulting structures often possess highly selective molecular recognition sites that would be difficult to access using traditional covalent bonding, a feature that has contributed to a growing interest in employing these architectures as selective sensing platforms, 4d-f as well as new types of biological materials. 4l, 5 In this regard, MIMs comprised of macrocycles with radial pi-conjugation could provide new topological landscapes for a variety of applications owing to the exotic optical and electronic properties of structures possessing radial pi-conjugation. 3 Only very recently have the first of these fascinating interlocked structures appeared in the literature via passive template synthesis approaches. 6 Alternatively, the active-template method (AT) 4b, c could be a more powerful approach to these types of MIMs in that it does not rely on formation of a thermodynamically stable, preorganized complex and therefore a wider array of structures are possible. Specifically, in the AT approach, a metal bound to the macrocyclic component catalyzes bond formation in the interior of the structure which gives rise to the interlocked molecules. Herein, we report the syntheses of nanohoop macrocycles that, via an embedded 2,6-pyridyl moiety, participate in AT reactions, ultimately giving rise to a new class of highly compact, fluorescent [2]rotaxanes (Figure 1c). Moreover, we show that triazole-embedded nanohoop [2]rotaxanes coordinate metals in a reversible manner which is accompanied by drastic changes in the emission of the macrocyclic fluorophore. Motivated by these findings, we then describe the design and preparation of a self-immolative type of rotaxane sensing platform, ultimately highlighting key conceptual advantages of forming mechanical bonds with fluorescent carbon nanohoops via AT syntheses.</p><!><p>Ligand and rotaxane synthesis. Inspired by the work of Leigh and Goldup, 4b, g we initially considered leveraging the coordination ability of our previously reported bipyridyl-embedded nanohoops 3c in AT reactions to prepare mechanically-interlocked nanohoop structures. However, in contrast to macrocycles typically used in AT rotaxane synthesis, a catalytic metal bound to these conformationally rigid nanohoops would direct bond formation to the exterior of the macrocycle. 3c Thus, our investigations began by first preparing a nanohoop ligand that would direct bond-formation to the interior of the macrocyclic cavity, which we envisioned occurring through the incorporation of a single 2,6-pyridine unit into the nanohoop backbone. Based on the well-established reductive aromatization approach to nanohoop molecules, 3 "curved" diboronate Suzuki-Miyaura cross-coupling partner 1 (Figure 2) was first prepared. 3j With boronate 1 in hand, we next explored macrocyclization conditions where under dilute (5 mM) Suzuki-Miyaura cross-coupling conditions with commercially available 2,6-dibromo pyridine, the desired macrocycle 2 was isolated in excellent yield. With macrocycle 2 in hand, a H2SnCl4-based aromatization was pursued, which after deprotection with tetrabutylammonium fluoride (TBAF), the target macrocycle 3 was accessed in a 94% yield. Under similar conditions (See Supporting Figure 1), larger macrocycle 4 (Figure 2b) was also synthetically accessible (confirmed via X-ray crystallography, Figure 2c), albeit in slightly reduced yield. Importantly, in stark contrast to our previously reported pyridyl-nanohoops, these macrocycles now have a coordination moiety located on the interior of the macrocyclic cavity as confirmed via X-ray crystallography (Figure 2c). Additionally, as a consequence of breaking molecular orbital symmetry, 3k it should be noted that both 3 and 4 were fluorescent, with 3 emitting at λmax of 509 nm (φF = 0.14) and 4 at λmax of 476 nm (φF = 0.62). Of note, the all para substituted nanohoop with a single embedded pyridine is non-emissive; 3b a detailed investigation of the result of symmetry breaking on nanohoop fluorescence is disclosed in a separate study. 3o We next explored the potential of macrocycles 3 and 4 to participate in AT reaction. As a starting point (Figure 3), we first investigated the well-studied AT Cadiot-Chodkiewicz (AT-CC) reaction. 4h, i Based on the observed cavity size of macrocycle 4 in the solid-state (~10 Å, Figure 2c), trityl-stoppered alkyne 5a and tritylstoppered bromo-alkyne 6a were prepared via literature procedures. 4j After optimization, we found that when 1.2 equivalents of alkyne 5a and bromo-alkyne 6a were subjected to the AT-CC conditions in Figure 3, the desired [2]rotaxane 7a was isolated in 51% yield. We next tested the ability of smaller macrocycle 3 to participate in an AT-CC reaction, where it was found that the desired rotaxane 7b formed with no loss in reaction efficiency. While small macrocycles have been explored in some cases, 4g, 4i the rigid, shape-persistent nature of macrocycle 3 results in a calculated cavity size of only 7.8 Å (see Supporting Figure 5), rendering the formation of 7b striking. Based on this result, we were curious if rotaxanes could be prepared using simple, low-molecular weight stopper groups, which would broaden the types of structures that could be accessed. Accordingly, it was found that under identical conditions, 3,5-di-t-butyl-stoppered rotaxane 7c was readily formed, whereas unsubstituted benzene rings did not lead to rotaxane formation. Initial structural confirmation of each new [2]rotaxane was acquired via mass spectrometry (See Supporting Figures 2 and 3). In addition, as is typical with MIMs, multiple upfield NMR shifts were observed in the thread component of each suspected [2]rotaxane, which further suggested a mechanically interlocked structure. For example, proton Ha (Figure 3a for label) of the encircled thread in [2]rotaxanes 7a and 7b is shifted significantly upfield by nearly 1.5 ppm and 2.0 ppm, respectively, relative to free thread. 4k Owing to the unusually small nature of macrocycle 3, we were particularly interested in the structural features of [2]rotaxanes 7b and 7c. Indeed, turning to single crystal X-ray crystallography, the highly congested nature of these pi-rich rotaxanes was revealed, where it was found that the cavity of macrocycle 3 is nearly a perfect fit for the diyne thread units of both 7b and 7c (Figure 3b, c). In the case of 7b, the observed distance between the threaded diyne component and macrocycle 3 was between 3.8 and 4.1 Å (Figure 3b). Additionally, no solvent molecules were observed in either structure, consistent with a tightly packed crystal structure overall. Given that conjugated macrocycles have recently emerged as strong candidates for new types of electronic materials, 3m, 3l this result is particularly encouraging, as the ability to thread pi-conjugated fragments in close contact could lead to new geometric designs for electron/hole transporting materials.</p><p>Owing to the wide range of commercially-available azide and alkyne functionalized starting materials, we next pursued an active template Cu(I)-catalyzed azidealkyne cycloaddition (AT-CuAAC) reaction. 4b As a first pass, macrocycle 4 was subjected to conditions (Figure 4a) similar to those reported by Leigh et al., 4b which provided the corresponding trityl-stoppered rotaxane 9a in excellent yield. However, when macrocycle 3 was subjected to these conditions using 3,5-dimethyl ester substituted coupling partners (Figure 4a, 8c and 8b), the desired rotaxane 9b was formed in 14% yield. After screening various conditions, we found that the addition of acetic acid as well as elevated temperatures (100 o C via microwave irradiation) improved the yield considerably, with rotaxane 9b being isolated in 45% yield (Figure 4a, entry 2). While a more detailed investigation of the scope is ongoing, for our work presented here, we viewed [2]rotaxane 9b as a particularly versatile intermediate. For example, motivated by our recent report on the biocompatibility of nanohoops 3i as well the emerging interest in using fluorescent rotaxane architectures in biological environments, 5 9b was then saponified to access water-soluble [2]rotaxane 9c (Figure 4b), which retained the corresponding emission properties (φF = 0.07) in aqueous conditions. Additionally, outside of biological systems, carboxylate functionalized [2]rotaxanes have also recently been incorporated into metal-organic frameworks, 7 suggesting that this intermediate could act as a new pi-rich building block for unusual types of coordination polymers. Ultimately, these results highlight the generality of both the AT-CC and AT-CuAAC reactions, a feature that is expected to provide a variety of new types of pi-rich interlocked molecules and materials. Additionally, the narrow cavity of the nanohoops reduce the requirements of the stopper groups that can be employed, furthering the utility of these systems.</p><!><p>Mechanically interlocked molecules bearing nitrogen heteroatoms often engage in a wide range of unique coordination chemistry where, depending on the metal and resulting coordination geometry, features such as molecular shuttling, switching, and sensing can be enabled. 4 Thus, we were curious if the congested binding pocket of triazole [2]rotaxanes such as 9b could still participate in metal-coordination. To probe this, we next investigated the effects of metalation on 9b in both the solid-state and solution through single-crystal X-ray crystallography, 1 H NMR titrations, and fluorescence spectroscopy. It was found that without metal, in both the solid-state (Figure 5a) and solution (via an upfield resonance at -1.43 ppm), the macrocycle of 9b resides primarily over the propyl chain. However, in the presence of Cu(I), the macrocycle is now located on the opposite face  of the triazole unit (Figure 5b). A similar shuttling effect was observed when 9b was titrated with [Pd(MeCN)4](BF4)2. Notably, in presence of Pd(II), the resulting 1 H NMR spectrum was noticeably sharper relative to that observed with [Cu(MeCN)4](PF6) indicating a less dynamic system. Taken together, it can be concluded that, despite the sterically congested environment, triazole embedded rotaxanes bearing macrocycle 3 bind metals such as Cu(I) and Pd(II), with Pd(II) acting as a stronger binding metal. From these initial studies, an additional key result was observed; as the equivalents of Pd(II) increased, the fluorescence of 9b decreased. As can be seen in Figure 5c, the addition of 1.0 equivalent of [Pd(MeCN)4](BF4)2 to 9b results in a non-emissive rotaxane, i.e. Pd(II)-9b. Given that metalation with Pd(II) effectively traps the fluorophore in a non-emissive state, we expected that demetallation should result in a turn-on response. To validate this, non-emissive Pd(II)-9b was treated with 1.0 equivalent of ethylenediaminetetraacetic acid (EDTA), which quickly resulted in a pronounced 30 fold increase in emission (Figure 5c). Encouraged by this result, we were then curious if the emission of watersoluble rotaxane 9c could be modulated in a similar fashion. Indeed, as shown in Figure 5d, the emission of carboxylate rotaxane 9c was readily quenched in aqueous media (PBS buffer) via the addition of 1.0 equivalent of [Pd(MeCN)4](BF4)2 to give Pd(II)-9c. Upon addition of EDTA to this aqueous solution, it was again found that the emission quickly returned, albeit with reduced intensity (10 fold increase). These results suggest that rotaxanes such as 9b and 9c are promising candidates for the development of new metal-based sensing platforms in both organic and, perhaps more importantly, aqueous media. Furthermore, we anticipate that through modifications to the macrocycle and/or thread component, metal/analyte-specificity can be imparted and consequently reported via bright fluorescence. [2]rotaxane. Inspired by the fluorescence quenching results with metals, we envisioned a perhaps more general sensing platform in which a suitable thread component could serve as both a stopper and fluorescence quencher. Analyte induced bond cleavage of the thread could then perhaps release the quencher and lead to a turn-on fluorescence response. Encouraged by the ability of electron-deficient C60 to quench the emission of [10]CPP, 3l we hypothesized that macrocycle 3 could temporarily be rendered non-emissive through the incorporation of a cleavable, mechanically bound electron-deficient stopper moiety. To investigate this concept, we prepared a 3,5-dinitro functionalized rotaxane (9d) bearing a fluoride-cleavable triisopropylsilyl (TIPS) stopper group (Figure 6a, b) via our AT-CC conditions (See Supporting Figure 1b for details) which we found to be non-emissive (Figure 6c). Importantly, treatment of rotaxane 9d with 1.0 equivalent of tetra-nbutylammonium fluoride (TBAF) resulted in a nearly instantaneous dethreading event (Figura 6a), which was accompanied by a dramatic 123-fold increase in emission intensity, effectively serving as proof-of-concept for a highly responsive self-immolative rotaxane sensor. Additionally, through density functional theorem (DFT), it was found that the frontier molecular orbitals are redistributed relative to macrocycle 3 (Figure 6d). Specifically, for macrocycle 3 (See Supporting Figure 6), both the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) reside over the nanohoop backbone. In contrast, in the case of 9d, the HOMO is localized on the macrocyclic component and the LUMO is localized over the electron-deficient nitrobenzene stopper, consistent with a charge-transfer quenching mechanism. These results highlight the importance of electronic structure in the design of new nonemissive rotaxanes such as 9d and we expect that through a computationally guided design approach, a wide range of new analyte-selective, turn-on scaffolds can be prepared. For example, the analyte recognition site (TIPS stopper) of 9d can likely be replaced by a range of small, yet effective analyte-sensitive stopper groups such as boronates, amides, and esters-a key advantage of using a rigid macrocycle with small diameter. Moreover, we anticipate that this design can be applied to our larger mac-  rocycle, 4, as a means to access different emission wavelength and higher quantum yield. Work is currently underway toward the preparation and application of these structures and will be reported in due course.</p><!><p>In conclusion, this work demonstrates that by embedding a 2,6-pyridine coordination motif into the backbone of a nanohoop macrocycle, a range of diverse [2]rotaxanes are accessible via two different Cu(I)catalyzed active-metal template reactions. Based on fundamental metal coordination experiments with triazoleembedded [2]rotaxanes formed via AT-CuAAC reactions, we have found that two metals, Cu(I) and Pd(II), effectively shuttle the nanohoop macrocycle along the length of a triazole-functionalized thread. A key result from these studies was the observation that in both organic and aqueous conditions, metal-binding is easily monitored via dramatic changes in fluorescence emission. Expanding on this finding, we then designed and prepared, via an AT-CC reaction, a metal-free, non-emissive nanohoop [2]rotaxane, that has been stoppered with a fluorescence quenching 3,5-dinitrobenzyl unit and fluoride cleavable TIPS group. On bond cleavage, the quenching moiety is no longer in close proximity to the fluorophore, which results in a dramatic 123-fold increase in emission intensity, effectively serving as proof-of-concept for nanohoop rotaxane turn-on fluorescence sensors. Particularly noteworthy is that this self-immolative nanohoop [2]rotaxane was constructed in a modular fashion and therefore should be easily adaptable to other types of analytes and applications. More broadly, we expect that this active template synthetic strategy will provide efficient pathways for generating a wide array of very compact pirich interlocked molecules as well as materials. Finally, due to the highly tunable and rigid pi-rich pores, catalytic metals bound to the interior of nanohoops represent an intriguing area of future exploration.</p><!><p>Full experimental details and characterization data for compounds synthesized. All experimental details and titration experiments. All experimental details for crystallography data. This material is available free of charge via the Internet at http://pubs.acs.org.</p><!><p>* rjasti@uoregon.edu</p>