Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Abstract

•Design and synthesis of a supramolecular rotor within a porous Zn–porphyrinic cage•High-yielding IEDDA reaction enables facile construction of the rotor inside the cage•Initiation of both rotary and tumbling motions of the rotor with chemical stimuli•Reversible control of the dual mechanical motions by a photo-dissociable ligand The confinement of biological molecular machines, which are controlled by various chemical or physical stimuli within the cytoskeleton matrix, endows them with remarkable precision and programmable mechanical motions. Biological molecular machines are far more complex than any artificial molecular machine ever built. To mimic the intricate behavior of biological machinery, strategies need to be developed for embedding and confining artificial molecular machines within nanoscopic domains in which their functions can be specifically controlled by external stimuli. One such approach is the confinement of molecular machines inside porous organic cages. The high porosity of the organic cages provides ample room for uninterrupted movement, and its shape-persistent and interactive framework provides a high degree of control over the mechanical motion triggered by external stimuli. Our strategy may lay the foundation for the development of tunable molecular devices using porous organic cages. The confinement of molecular machines into nanostructured cages and controlling their functions by external stimuli holds great potential for the creation of smart functional materials that imitate the embodied intelligence of biological processes. Herein, we report the construction of a supramolecular rotor in a porous Zn-metallated porphyrinic cage (1) by encapsulation of a tetrazine-based linear axle (LA) via metal-ligand coordination bond, followed by post-assembly modification to append a controllable side arm to LA via inverse electron demand Diels-Alder (IEDDA) reaction. While the rotor alone shows nearly no motion, the addition of pyridine derivatives as a zinc coordinating ligand results in both 90° jump-like rotary motion of the rotor and slow tumbling motion of the rotor axle in a stochastic manner. Interestingly, the dual motions of the rotor can be reversibly controlled by the UV and visible light-induced coordination and dissociation of an azopyridine-based ligand with Zn centers as a signal transducer. The confinement of molecular machines into nanostructured cages and controlling their functions by external stimuli holds great potential for the creation of smart functional materials that imitate the embodied intelligence of biological processes. Herein, we report the construction of a supramolecular rotor in a porous Zn-metallated porphyrinic cage (1) by encapsulation of a tetrazine-based linear axle (LA) via metal-ligand coordination bond, followed by post-assembly modification to append a controllable side arm to LA via inverse electron demand Diels-Alder (IEDDA) reaction. While the rotor alone shows nearly no motion, the addition of pyridine derivatives as a zinc coordinating ligand results in both 90° jump-like rotary motion of the rotor and slow tumbling motion of the rotor axle in a stochastic manner. Interestingly, the dual motions of the rotor can be reversibly controlled by the UV and visible light-induced coordination and dissociation of an azopyridine-based ligand with Zn centers as a signal transducer. “Confinement effect” at various length scales is believed to play an indispensable role in evolution of life,1Szostak J.W. Bartel D.P. Luisi P.L. Synthesizing life.Nature. 2001; 409: 387-390Google Scholar,2Hansma H.G. The power of crowding for the origins of life.Orig. Life Evol. Biosph. 2014; 44: 307-311Google Scholar stabilization of biomolecules,3Zhou H.-X. Dill K.A. Stabilization of proteins in confined spaces.Biochemistry. 2001; 40: 11289-11293Google Scholar,4Panganiban B. Qiao B. Jiang T. DelRe C. Obadia M.M. Nguyen T.D. Smith A.A.A. Hall A. Sit I. Crosby M.G. et al.Random heteropolymers preserve protein function in foreign environments.Science. 2018; 359: 1239-1243Google Scholar and in controlling the reactivity and functions of chemical systems.5Grommet A.B. Feller M. Klajn R. Chemical reactivity under nanoconfinement.Nat. Nanotechnol. 2020; 15: 256-271Google Scholar, 6Rebek J. Molecular behavior in small spaces.Acc. Chem. Res. 2009; 42: 1660-1668Google Scholar, 7Yoshizawa M. Klosterman J.K. Fujita M. Functional molecular flasks: new properties and reactions within discrete, self-assembled hosts.Angew. Chem. Int. Ed. Engl. 2009; 48: 3418-3438Google Scholar The physical and chemical properties of a molecule can be astutely altered once confined within a suitable nanoscopic domain.6Rebek J. Molecular behavior in small spaces.Acc. Chem. Res. 2009; 42: 1660-1668Google Scholar,7Yoshizawa M. Klosterman J.K. Fujita M. Functional molecular flasks: new properties and reactions within discrete, self-assembled hosts.Angew. Chem. Int. Ed. Engl. 2009; 48: 3418-3438Google Scholar Among other complex macromolecular or supramolecular systems, the confinement of biological machineries within specific nanoscopic domains inside the cellular matrix, which provides an environment that is electronically and sterically different from the extracellular matrix, is the very basis for their remarkable precision and programmed mechanical motion of their constituent components and associated functions.8Kinbara K. Aida T. Toward intelligent molecular machines: directed motions of biological and artificial molecules and assemblies.Chem. Rev. 2005; 105: 1377-1400Google Scholar, 9Schliwa M. Woehlke G. Molecular motors.Nature. 2003; 422: 759-765Google Scholar, 10Vale R.D. Milligan R.A. The way things move: looking under the hood of molecular motor proteins.Science. 2000; 288: 88-95Google Scholar Most of these biomolecular machines are controlled by various chemical or physical stimuli and transform their energy to perform a specific biological function.8Kinbara K. Aida T. Toward intelligent molecular machines: directed motions of biological and artificial molecules and assemblies.Chem. Rev. 2005; 105: 1377-1400Google Scholar One of the most celebrated examples is adenosine triphosphate (ATP) synthase, where the rotatory motion of the enzyme subunits is controlled by chemical processes, such as proton (or Na+) flux or ATP hydrolysis.11Stock D. Leslie A.G. Walker J.E. Molecular architecture of the rotary motor in ATP synthase.Science. 1999; 286: 1700-1705Google Scholar,12Noji H. Yasuda R. Yoshida M. Kinosita K. Direct observation of the rotation of F1-ATPase.Nature. 1997; 386: 299-302Google Scholar In some cases, transmembrane proteins remotely control the mechanical motion of biomolecules, such as bacteriorhodopsin or halorhodopsin in light-driven ion pumps.13Jia Y. Li J. Reconstitution of FoF1-ATPase-based biomimetic systems.Nat. Rev. Chem. 2019; 3: 361-374Google Scholar, 14Lanyi J.K. Pohorille A. Proton pumps: mechanism of action and applications.Trends Biotechnol. 2001; 19: 140-144Google Scholar, 15Richard P. Pitard B. Rigaud J.-L. ATP synthesis by the FoF1-ATPase from the thermophilic Bacillus PS3 co-reconstituted with bacteriorhodopsin into liposomes. Evidence for stimulation of ATP synthesis by ATP bound to a noncatalytic binding site.J. Biol. Chem. 1995; 270: 21571-21578Google Scholar Inspired by such natural systems, numerous stimuli-controlled artificial molecular machines have been developed.16Kottas G.S. Clarke L.I. Horinek D. Michl J. Artificial molecular rotors.Chem. Rev. 2005; 105: 1281-1376Google Scholar, 17Erbas-Cakmak S. Leigh D.A. McTernan C.T. Nussbaumer A.L. Artificial molecular machines.Chem. Rev. 2015; 115: 10081-10206Google Scholar, 18Kassem S. van Leeuwen T. Lubbe A.S. Wilson M.R. Feringa B.L. Leigh D.A. Artificial molecular motors.Chem. Soc. Rev. 2017; 46: 2592-2621Google Scholar, 19Baroncini M. Silvi S. Credi A. Photo- and redox-driven artificial molecular motors.Chem. Rev. 2020; 120: 200-268Google Scholar, 20Coskun A. Banaszak M. Astumian R.D. Stoddart J.F. Grzybowski B.A. Great expectations: can artificial molecular machines deliver on their promise?.Chem. Soc. Rev. 2012; 41: 19-30Google Scholar, 21Astumian R.D. Kay E.R. Leigh D.A. Zerbetto F. Design principles for Brownian molecular machines: how to swim in molasses and walk in a hurricane.Proc. Natl. Acad. Sci. USA. 2006; 46: 10771-10776Google Scholar One approach to further explore the potential of artificial molecular machines for practical applications and to gain a better control over their functions is to confine them inside a 3D nanoscopic environment.22Wilson B.H. Vojvodin C.S. Gholami G. Abdulla L.M. O’Keefe C.A. Schurko R.W. Loeb S.J. Precise spatial arrangement and interaction between two different mobile components in a metal-organic framework.Chem. 2021; 7: 202-211Google Scholar, 23Su Y.-S. Lamb E.S. Liepuoniute I. Chronister A. Stanton A.L. Guzman P. Pérez-Estrada S. Chang T.Y. Houk K.N. Garcia-Garibay M.A. Brown S.E. Dipolar order in an amphidynamic crystalline metal-organic framework through reorienting linkers.Nat. Chem. 2021; 13: 278-283Google Scholar, 24Danowski W. van Leeuwen T. Abdolahzadeh S. Roke D. Browne W.R. Wezenberg S.J. Feringa B.L. Unidirectional rotary motion in a metal-organic framework.Nat. Nanotechnol. 2019; 14: 488-494Google Scholar, 25Orlova T. Lancia F. Loussert C. Iamsaard S. Katsonis N. Brasselet E. Revolving supramolecular chiral structures powered by light in nanomotor-doped liquid crystals.Nat. Nanotechnol. 2018; 13: 304-308Google Scholar, 26Kaleta J. Chen J. Bastien G. Dračínský M. Mašát M. Rogers C.T. Feringa B.L. Michl J. Surface inclusion of unidirectional molecular motors in hexagonal Tris(o-phenylene)cyclotriphosphazene.J. Am. Chem. Soc. 2017; 139: 10486-10498Google Scholar, 27Li Q. Fuks G. Moulin E. Maaloum M. Rawiso M. Kulic I. Foy J.T. Giuseppone N. Macroscopic contraction of a gel induced by the integrated motion of light-driven molecular motors.Nat. Nanotechnol. 2015; 10: 161-165Google Scholar, 28Seo J. Matsuda R. Sakamoto H. Bonneau C. Kitagawa S. A pillared-layer coordination polymer with a rotatable pillar acting as a molecular gate for guest molecules.J. Am. Chem. Soc. 2009; 131: 12792-12800Google Scholar These nanoporous confinements can serve as a stable scaffold to anchor the molecular machines in a specific orientation so that a collective effect of the functioning of individual machines can be harnessed to execute a predetermined function, a long-cherished goal in the area of amphidynamic materials.29Roy I. Stoddart J.F. Amphidynamic crystals key to artificial molecular machines.Trends Chem. 2019; 1: 627-629Google Scholar, 30Colin-Molina A. Karothu D.P. Jellen M.J. Toscano R.A. Garcia-Garibay M.A. Naumov P. Rodríguez-Molina B. Thermosalient amphidynamic molecular machines: motion at the molecular and macroscopic scales.Matter. 2019; 1: 1033-1046Google Scholar, 31Karlen S.D. Garcia-Garibay M.A. Amphidynamic crystals: structural blueprints for molecular machines.in: Kelly T.R. Molecular Machines. Springer, 2005: 179-227Google Scholar Nevertheless, there always exists a tradeoff between controlling the orientation of the machines through rigid confinement and maintaining the required flexibility to carry out the specific motion as efficiently as in the solution state. In this regard, porous molecular cages are particularly interesting for encapsulating guest molecules as molecular machines via non-covalent interactions and to study the effect of molecular confinement on their functions. Due to their accessible internal cavities for nanoscale confinement, shape persistence, and their molecular solubility, the molecular machines can retain their solution-state efficiency even after being confined inside the cages.32Rizzuto F.J. Ramsay W.J. Nitschke J.R. Otherwise unstable structures self-assemble in the cavities of cuboctahedral coordination cages.J. Am. Chem. Soc. 2018; 140: 11502-11509Google Scholar, 33Löffler S. Lübben J. Wuttke A. Mata R.A. John M. Dittrich B. Clever G.H. Internal dynamics and guest binding of a sterically overcrowded host.Chem. Sci. 2016; 7: 4676-4684Google Scholar, 34Mugridge J.S. Szigethy G. Bergman R.G. Raymond K.N. Encapsulated Guest-Host dynamics: guest rotational barriers and tumbling as a probe of host interior cavity space.J. Am. Chem. Soc. 2010; 132: 16256-16264Google Scholar, 35Kitagawa H. Kobori Y. Yamanaka M. Yoza K. Kobayashi K. Encapsulated-guest rotation in a self-assembled heterocapsule directed toward a supramolecular gyroscope.Proc. Natl. Acad. Sci. USA. 2009; 106: 10444-10448Google Scholar Such an alliance of molecular machines and porous organic cages can, in principle, serve as a functional nanoscale unit for the controlled manipulation (via catalysis) and transport of other chemical species (such as ions).36Elemans J.A.A.W. Nolte R.J.M. Porphyrin cage compounds based on glycoluril—from enzyme mimics to functional molecular machines.Chem. Commun. 2019; 55: 9590-9605Google Scholar,37Gilissen P.J. White P.B. Berrocal J.A. Vanthuyne N. Rutjes F.P.J.T. Feringa B.L. Elemans J.A.A.W. Nolte R.J.M. Molecular motor-functionalized porphyrin macrocycles.Nat. Commun. 2020; 11: 5291Google Scholar Despite these inherent advantages, it still remains synthetically challenging to install molecular machines inside the nano-sized porous organic cages and to control the molecular motion by chemical or/and physical stimuli. Of particular interest is controlling confined molecular machines remotely by an external stimulus, which may unlock new properties of these systems with spatiotemporal precision. In comparison with other remotely controlled stimuli, light has unique advantages of being a clean energy source and having the ability to be transiently delivered at a precise location in a solution.38van Leeuwen T. Lubbe A.S. Štacko P. Wezenberg S.J. Feringa B.L. Dynamic control of function by light-driven molecular motors.Nat. Chem. Rev. 2017; 1: 96Google Scholar,39Kundu P.K. Samanta D. Leizrowice R. Margulis B. Zhao H. Börner M. Udayabhaskararao T. Manna D. Klajn R. Light-controlled self-assembly of non-photoresponsive nanoparticles.Nat. Chem. 2015; 7: 646-652Google Scholar These benefits have led to the development of a large number of photoresponsive molecular machines, self-assemblies, smart materials, drug delivery systems, etc.40Ube H. Yasuda Y. Sato H. Shionoya M. Metal-centred azaphosphatriptycene gear with a photo- and thermally driven mechanical switching function based on coordination isomerism.Nat. Commun. 2017; 8: 14296Google Scholar, 41Qu D.H. Wang Q.C. Zhang Q.W. Ma X. Tian H. Photoresponsive host-guest functional systems.Chem. Rev. 2015; 115: 7543-7588Google Scholar, 42Klajn R. Spiropyran-based dynamic materials.Chem. Soc. Rev. 2014; 43: 148-184Google Scholar, 43Russew M.M. Hecht S. Photoswitches: from molecules to materials.Adv. Mater. 2010; 22: 3348-3360Google Scholar Usually, a molecular machine consists of a light-responsive component, such as azobenzenes,44Moosa B. Alimi L.O. Shkurenko A. Fakim A. Bhatt P.M. Zhang G. Eddaoudi M. Khashab N.M. A polymorphic azobenzene cage for energy-efficient and highly selective p-xylene separation.Angew. Chem. Int. Ed. Engl. 2020; 59: 21367-21371Google Scholar,45Wang Z. Knebel A. Grosjean S. Wagner D. Bräse S. Wöll C. Caro J. Heinke L. Tunable molecular separation by nanoporous membranes.Nat. Commun. 2016; 7: 13872Google Scholar dithienylethenes,46Zheng Y. Sato H. Wu P. Jeon H.J. Matsuda R. Kitagawa S. Flexible interlocked porous frameworks allow quantitative photoisomerization in a crystalline solid.Nat. Commun. 2017; 8: 100Google Scholar,47Li R.J. Tessarolo J. Lee H. Clever G.H. Multi-stimuli control over assembly and guest binding in metallosupramolecular hosts based on dithienylethene photoswitches.J. Am. Chem. Soc. 2021; 143: 3865-3873Google Scholar spiropyranes,48Williams D.E. Martin C.R. Dolgopolova E.A. Swifton A. Godfrey D.C. Ejegbavwo O.A. Pellechia P.J. Smith M.D. Shustova N.B. Flipping the switch: fast photoisomerization in a confined environment.J. Am. Chem. Soc. 2018; 140: 7611-7622Google Scholar, 49Mondal B. Ghosh A.K. Mukherjee P.S. Reversible multi-stimuli switching of a spiropyran-functionalized organic cage in solid and solution.J. Org. Chem. 2017; 82: 7783-7790Google Scholar, 50Kundu P.K. Olsen G.L. Kiss V. Klajn R. Nanoporous frameworks exhibiting multiple stimuli responsiveness.Nat. Commun. 2014; 5: 3588Google Scholar etc.,51Castiglioni F. Danowski W. Perego J. Leung K.-C.F. Sozzani P. Bracco S. Wezenberg S.J. Comotti A. Feringa B.L. Modulation of porosity in a solid material enabled by bulk photoisomerization of an overcrowded alkene.Nat. Chem. 2020; 12: 595-602Google Scholar to control its mechanical motion by light in the confinement. However, using a light source to control a molecular machine that is devoid of any photoresponsive component can be very interesting considering its resemblance with the biological machines, e.g., light-driven ion pumps. We have recently reported cube-shaped porous organic cages self-assembled from six square-shaped porphyrins and eight triangular linkers by dynamic covalent chemistry.52Mukhopadhyay R.D. Kim Y. Koo J. Kim K. Porphyrin boxes.Acc. Chem. Res. 2018; 51: 2730-2738Google Scholar, 53Benke B.P. Aich P. Kim Y. Kim K.L. Rohman M.R. Hong S. Hwang I.-C. Lee E.H. Roh J.H. Kim K. Iodide-selective synthetic ion channels based on shape-persistent organic cages.J. Am. Chem. Soc. 2017; 139: 7432-7435Google Scholar, 54Hong S. Rohman M.R. Jia J. Kim Y. Moon D. Kim Y. Ko Y.H. Lee E. Kim K. Porphyrin boxes: rationally designed porous organic cages.Angew. Chem. Int. Ed. Engl. 2015; 54: 13241-13244Google Scholar Shape-persistent porphyrinic cages, due to their well-defined receptor cavities, are ideal platforms to install molecular machines in a precise and predictable manner. Previously, a number of Zn–porphyrins-based dynamic molecular machines have been reported.55Goswami A. Schmittel M. Double rotors with fluxional axles: domino rotation and azide–alkyne Huisgen cycloaddition catalysis.Angew. Chem. Int. Ed. Engl. 2020; 59: 12362-12366Google Scholar, 56Gaikwad S. Goswami A. De S. Schmittel M. A Metalloregulated four-state nanoswitch controls two-step sequential catalysis in an eleven-component system.Angew. Chem. Int. Ed. Engl. 2016; 55: 10512-10517Google Scholar, 57Liu S. Kondratuk D.V. Rousseaux S.A.L. Gil-Ramírez G. O'Sullivan M.C. Cremers J. Claridge T.D.W. Anderson H.L. Caterpillar track complexes in template-directed synthesis and correlated molecular motion.Angew. Chem. Int. Ed. Engl. 2015; 54: 5355-5359Google Scholar, 58Muraoka T. Kinbara K. Aida T. Mechanical twisting of a guest by a photoresponsive host.Nature. 2006; 440: 512-515Google Scholar However, the establishment of a rational and high-yielding synthetic strategy to precisely organize molecular machines inside Zn–porphyrin-based porous organic cages with a 3D encompassed intrinsic nanoscopic cavity and to control their functions with multiple external stimuli requires further exploration. Herein, we report the synthesis of a supramolecular rotor built inside a porous Zn–porphyrinic cage (1) by insertion of a tetrazine-based LA followed by post-assembly modification to append a controllable side arm to the axle. The 1H and 2D EXSY NMR studies reveal a 90° jump-like random rotary motion of the rotor arm and a slow tumbling motion of the rotor axle upon the addition of pyridine derivatives, which compete with the rotor for zinc–porphyrin coordination. Computational calculations confirm the stochastic nature of the molecular motions. Moreover, the rotation rates of these two motions can be adjusted by varying the amounts of pyridine derivatives. The reversible control over the dual motions of the rotor is achieved by UV and visible light-induced coordination and dissociation of azopyridine-based photo-dissociable ligand (PDL), which acts as a signal transducer. The nanoscale hydrophobic cavity of the Zn–porphyrinic cage and the remotely controllable functions of the rotor may collectively lead to the development of tunable molecular devices with potential applications in reversible on-off ion channels, switchable catalysis, and drug delivery. At the outset of this work, we synthesized a pyridine terminated LA (length, ∼1.9 nm, see synthesis in Scheme S1), which can coordinate to two Zn metal centers of 1 (distance, ∼2.3 nm), across the cavity (Figure S1). LA consists of a central tetrazine moiety that can react efficiently with alkenes or alkynes dienophiles via inverse electron demand Diels-Alder (IEDDA) reaction to attach a new functionality on LA. By using the remarkably high-yielding IEDDA reactions for post-assembly modification, a number of different rotor moieties can be precisely incorporated into the cage. We envisaged that post-assembly modification of LA inside 1 with (1R,8S,9s)-bicyclo[6.1.0]-non-4-yn-9-ylmethanol (L1) may provide a controllable side arm for the freely rotating axle as L1 contains a hydroxy group (–OH) at the terminal position, which can coordinate with the “equatorial” Zn–porphyrins of 1 (Figure 1). The mechanical motion of the resulting assembly L1-LA⊂1 can be triggered in the presence of chemical stimuli, such as pyridine derivatives, which can coordinate with the Zn–porphyrinic faces of L1-LA⊂1 from outside the hydrophobic cavity, thus initiating the motion by inhibiting the formation of coordination bonds between the Zn centers of 1 and the rotor L1-LA (Figure 1B). The axle insertion experiment was performed by adding a stoichiometric amount of LA to the chloroform solution of 1, yielding LA⊂1 (Figure 1A). The binding affinity (Ka) of 1 for LA was determined to be 3.8 ± 0.1 × 107 M−1 by UV-vis titration (Figure S2), confirming a strong host-guest interaction between 1 and LA. In the 1H NMR spectrum of LA⊂1 in toluene-d8, LA protons showed a large upfield shift compared with their free state, due to the strong ring current of the porphyrin units. In particular, the α-py protons that are closest to the porphyrin ring showed the largest upfield shift (8.84 to 1.88 ppm), indicating that LA is precisely positioned inside 1 (Figure S3). Moreover, the insertion of LA into the cavity of 1 splits the identical pyrrolic protons (a) of all six porphyrins of 1 into three sets: a1 protons for two “axial” porphyrins (connecting to the axle) and a2 and a3 protons for four “equatorial” porphyrins (parallel to the axle). The a2 and a3 protons show different chemical shifts as they are directed toward the axial and equatorial porphyrins, respectively (Figure 2). The NMR splitting pattern of the cage protons suggests the O → D4 desymmetrization of the cage structure. All peaks were assigned by several 2D NMR experiments (1H–13C HSQC [heteronuclear single quantum coherence spectroscopy], 1H–1H COSY [homonuclear correlation spectroscopy], and ROESY [rotating-frame nuclear overhauser effect spectroscopy]; Figures S4–S7). A single trace in the DOSY (diffusion-ordered spectroscopy) spectrum confirmed the presence of a single species (LA⊂1) in the solution (Figure S8). Finally, X-ray structure analysis of LA⊂1 (for crystallization conditions, see supplemental information) confirmed the coordination of the py moieties of LA to the Zn metal centers of the opposite faces of 1 (Zn–Npy = 2.081(5) Å). In the crystal structure, LA⊂1 maintained an overall cube-shape with the same opposite Zn⋅⋅⋅Zn distance (23.8 Å) in all three directions despite the insertion of LA (Figure 2A). To demonstrate facile post-assembly modification of LA⊂1, norbornadiene was chosen as a model compound for IEDDA reaction that quantitatively converts the tetrazine-based axle (LA) to a pyridazine-based axle (PLA) inside 1 (PLA⊂1) within 4 h at room temperature, which is manifested by the disappearance of 1H NMR signals of LA and the appearance of PLA signals (Figures S9 and S10). The formation of PLA⊂1 was confirmed by 2D NMR and DOSY analysis (Figures S11–S13). The post-assembly modification of LA⊂1 with L1 substrate (1 equiv per tetrazine) by IEDDA reaction proceeded almost instantly at room temperature to furnish a ring-fused pyridazine-based axle inside 1 (L1-LA⊂1), as verified by 1H NMR, MALDI-TOF, and elemental analysis (EA) (Figures S14 and S15). Thermal stability was confirmed by thermogravimetric analysis (TGA) (Figures S16 and S17). In the 1H NMR spectrum of L1-LA⊂1 in toluene-d8, the L1 protons are shifted in the upfield region (appeared between 0 to −5 ppm), which may be due to their close proximity with one of the “equatorial” Zn–porphyrin from inside the cavity. In addition, the pyrrolic protons a3 of the equatorial Zn–porphyrins of LA⊂1 are divided into two sets a3a and a3b, indicating further desymmetrization of the cage structure (Figure 2C). The splitting of phenyl and imine protons also suggests the lowering of cage symmetry. All peaks were assigned by several 2D NMR experiments (Figures S18–S21). Moreover, DOSY experiment confirmed the single species in solution (Figure S22). Unexpectedly, the pyrrolic protons a1 and a2 of L1-LA⊂1 showed no splitting, the reason for which is currently not fully understood. On the other hand, upon directly mixing the 1:1 ratio of L1-LA and 1, a non-symmetrical pattern of LA protons was observed in the 1H NMR spectrum, indicating that L1-LA coordinates to 1 from outside the cage in a monodentate fashion (Figure S23), probably due to small-sized windows of the cage and long alkyl chains blocking the cage windows. After numerous attempts, the structure of L1-LA⊂1 has been finally confirmed by single-crystal X-ray crystallography (Figure 2B). The molecular structure revealed that L1-LA resides inside 1 by the coordination of the py groups of L1-LA to the “axial” Zn–porphyrins (Zn–Npy = 2.129(5) Å) and the hydroxy group (–OH) to the “equatorial” Zn–porphyrin (Zn–OH = 2.379(5) Å). As shown in Figure 2B, the slightly bent axle LA (bent angle, ∼167°) and curved shape of the side arm L1 assist in the coordination of the hydroxy group (–OH) with one of the “equatorial” Zn–porphyrins. The opposite Zn⋅⋅⋅Zn distances in the a and b axes are both 23.7 Å, whereas that in the c axis is 20.1 Å. The shorter Zn⋅⋅⋅Zn distance may be ascribed to the coordination of the –OH group of L1-LA to the equatorial Zn–porphyrin. The bent shape of LA and the curved shape of L1 were also observed in the X-ray crystal structure of L1-LA (Figure S24). Next, we investigated the operation of the supramolecular rotor L1-LA⊂1. In the absence of external stimulus, upon cooling the solution of L1-LA⊂1 (in toluene-d8) down to 243 K, no splitting of pyrrolic protons peaks was observed in the 1H NMR spectrum (Figure S25), which suggests that L1-LA is either rotating too slowly on the NMR timescale or no rotation at all, possibly due to the Zn–OHL1-LA coordination bond. It is known that Zn–porphyrins bind more strongly to pyridine than to alcohols.59Olsson S. Dahlstrand C. Gogoll A. Design of oxophilic metalloporphyrins: an experimental and DFT study of methanol binding.Dalton Trans. 2018; 47: 11572-11585Google Scholar In fact, coordination of pyridine to the Zn center can replace the Zn–OH coordination bond for maintaining a five-coordinate binding mode. The coordination of pyridine to L1-LA⊂1 is borne out by the upfield shift of the pyridine protons peaks in the 1H NMR spectrum. However, in the presence of excess pyridine, the peaks shifted back to the downfield region due to the establishment of a fast equilibrium between coordinated and free pyridine molecules (Figure S26). Thereafter, the effect of the addition of the chemical stimulant (pyridine) to L1-LA⊂1 was studied in detail by 1H NMR spectroscopy. Upon stepwise addition of pyridine (up to 6 equiv) to L1-LA⊂1, the pyrrolic protons a3a and a3b of the four equatorial porphyrins merged into a sharp singlet at 8.98 ppm (Figure 3A). The imine protons and the phenyl protons that correspond to the equatorial porphyrins also merged, indicating the symmetrization of the cage framework (Figure S26A). Additionally, the side arm (L1) protons that appear between 0 to −5 ppm due to their close proximity with one of the equatorial Zn–porphyrins broadened and shifted slightly downfield (Figure S26B). The symmetrical structure of the cage framework as derived from the 1H NMR spectroscopic studies and broadened resonances of the side arm protons suggested that the coordination of pyridine molecules to the Zn centers dissociated the Zn–OH coordination bond, and, as a result, the side arm started jumping rapidly around all four equatorial Zn–porphyrin stations at room temperature by using the rotor axle as a hinge (Figure 1B). To confirm that the mixing of the peaks is due to the rotary motion and not to pyridine coordination, 1H NMR titration between pyridine (0–6 equiv) and 1 was performed, which showed that the coordination of pyridine does not affect the protons signals of cage 1 due to its relatively rapid association/dissociation equilibrium with the Zn–porphyrins of 1 on the NMR timescale (Figure S27). Further insight into the rotor dynamics was obtained from variable-temperature (VT) 1H NMR studies. While the a3a and a3b protons of L1-LA⊂1 appeared as a single peak in the presence of 6 equiv of pyridine at 298 K, the peak was broadened at 263 K and emerged as two distinct peaks at 9.05 and 9.09 ppm at 243 K (Figure 3B). The 1H NMR could not be recorded below 243 K due to poor solubility of L1-LA⊂1 in toluene-d8 at the low temperatures. Upon raising the temperature back to 298 K, the peaks again merged into a single peak at 8.99 ppm (Figure S28). These experiments further corroborated the notion that the rotor exhibits a fast rotary motion in the presence of the chemical stimulant pyridine, whereas the rotation decelerates at low temperatures. Line shape analysis of the VT 1H NMR data in the presence of 6 equi