This Outlook is intended to be part of a conversation that is already taking place at scientific meetings and in the pages of scientific journals; by providing concrete examples of the potential role of simplicity in future developments, we hope to contribute to these discussions. We should make clear that we are in no way attempting to suggest that the field should focus on application-driven research—progress has always and will always rely on the raw curiosity of outstanding scientists. However, where new applications are proposed for interlocked molecular machines, we propose simplicity as one parameter that can be used in evaluating and developing these claims.

This Outlook is intended to be part of a conversation that is already taking place at scientific meetings and in the pages of scientific journals; by providing concrete examples of the potential role of simplicity in future developments, we hope to contribute to these discussions.

However, despite the many impressive demonstrations of interlocked molecular machine prototypes, their use as real-world tools remains elusive. In this Outlook we would like to hold simplicity up as one parameter to be addressed for interlocked molecular machines to make the leap beyond the academic laboratory bench. Ironically “simplicity” is a relatively complex concept as it is both highly subjective and context dependent. Here, we identify three forms of simplicity that could be considered: simplicity of structure, simplicity of operation, and the balance between the overall simplicity of the system and the value of the application proposed. We illustrate each of these points with positive recent examples of molecular machine prototypes whose behavior and/properties could in theory have real-world applications, from stimuli responsive catalysts through to machines capable of the synthesis of sequence-controlled polymers.

In this Outlook we would like to hold simplicity up as one parameter to be addressed for interlocked molecular machines to make the leap beyond the academic laboratory bench.

Inspired at least in part by nature’s nanotechnology, for many years, scientists have worked to develop a corresponding artificial chemical nanotechnology. (8) Much of this work has focused on the use of mechanically interlocked molecules such as a rotaxanes and catenanes, (9) by taking advantage of the ability of the interlocked subcomponents to undergo large amplitude relative motion. Many motivations have been proposed for this effort including using minimalist models of natural machines to aid their analysis and understanding, (10) the development of an artificial molecular nanotechnology that rivals the systems found in nature in order to achieve lifelike functions (which has obvious echoes in synthetic biology) or to overcome existing chemical problems, and of course the simple aesthetic and scientific challenge of doing so—the “because it’s there” justification of mountaineers. Of these general motivations, the potential of a synthetic molecular nanotechnology to solve existing chemical challenges has become increasingly salient as the field has matured and particularly since the award of the Nobel Prize in 2016 to Feringa, (11) Sauvage, (12) and Stoddart, (13) the latter for their work on mechanically interlocked molecular machines. Indeed, the Nobel committee highlighted that, as with computing, “miniaturization of technology can lead to a revolution”. (14)

The molecular machines employed by nature almost seem the work of science fiction, especially when represented in stylized form with the chemical detail removed. (1) Using these molecular machines, living systems carry out many of the tasks essential for life including converting chemical energy from one form to another (ATP synthase), (2) moving large cargoes around within cells (kinesin), (3) replicating information-rich biopolymers (DNA synthase), (4) carrying out complex chemical synthesis (the ribosome), (5) and generating macroscopic movement (myosin). (6) Typically this is achieved by coupling chemical reactions and biased Brownian motion to achieve the desired task. (7)

In summary, although interlocked molecular machines are vastly simpler than their biological counterparts, there remains a significant need to optimize their production in the progression from prototype to application. This requires attention to all aspects of the synthesis, not just the mechanical bond forming step, and may include reverse engineering the required behavior into a minimalist structure, including replacing a bespoke, hard to access macrocycle in the prototype with a ring that is more readily available, or just “simply” optimizing the chemistry to ensure maximum yield and minimum steps: in short, the same problem that synthetic chemists working in a range of areas have been overcoming for decades.

Finally, perhaps the most striking example of a synthetically optimized and widely useful macrocycle is the pillararene macrocycles, introduced by Ogoshi and co-workers. (31) These can be made in excellent yield from extremely simple building blocks ( Figure 3 e) and form threaded structures due to a combination of dipole–dipole interactions and solvophobic effects. They can also be readily functionalized for a wide range of applications and thus are now extremely widely used, particularly in the context of supramolecular polymers, (32) although to date, less commonly as components of molecular machines.

The active template approach to interlocked molecules is, potentially, much more tolerant to modifications of the macrocycle structure than approaches based on thermodynamically stable complexes ( Figure 3 d), (28) and recent efforts have extended the principle from metal-mediated reactions to metal-free organocatalytic systems. (29) Bipyridine macrocycles (e.g.,) are particularly effective in this approach, and for this reason we recently developed an optimized, flexible synthesis of these building blocks that can be readily scaled to produce gram quantities of these useful starting materials. (30)

Finally, one returns to the synthetic chemist’s standard solution: invest effort in optimizing the synthesis of key macrocyclic building blocks that are particularly useful. (24) Simple crown ether-based macrocycles are a good example of this; metal-based templates have been employed very effectively to achieve high-yielding syntheses of some of these ubiquitous building blocks. (25) However, these systems do not tolerate significant structural modification in the metal binding region as the metal templated synthesis relies on these interactions, as does the subsequent mechanical bond forming step. Similarly, cucurbituril macrocycles are extremely effective in host–guest complex formation, (26) and their synthesis has now been streamlined significantly, (27) although, again, despite recent advances, functionalization of these macrocycles is challenging.

Alternatively, there are a small set of macrocycles that can be formedduring the mechanical bond formation from smaller, easily accessible fragments, removing the need to separately synthesize the macrocyclic component. Tetralactam macrocycles ( Figure 3 b) (22) popularized by Leigh, and Stoddart’s “blue box” rings ( Figure 3 c) (23) are excellent examples of these multicomponent approaches to interlocked molecule synthesis, and both have been extensively demonstrated as components of interlocked molecular machines. However, because specific noncovalent interactions are involved in kinetically favoring macrocycle formation, small structural modifications of the ring, that may be necessary to achieve a desired behavior or function in a more complex molecular device, can lead to a significant reduction in the yield of this key step. (22e)

Perhaps the best solution to the synthetic challenge of macrocycle synthesis is to focus on examples that are essentially “free” because they are produced by natural systems. Here, the only obvious examples are the cyclodextrins (CDs) ( Figure 3 a), which form threaded complexes due to solvophobic effects. (20) Native, unfunctionalized CDs are readily available at low cost in three sizes, and thus, if a complex molecular machine can be designed based on CDs, its synthesis is greatly simplified. However, although a great deal of elegant chemistry has been developed, (21) the synthesis of functionalized CD rings remains challenging, somewhat offsetting the benefits of the availability of the native CD ring itself.

However, even the simplest interlocked molecular shuttle runs up against a general synthetic problem; all interlocked molecular machines require at least one macrocycle. Indeed, the production of macrocycles suitable for inclusion in molecular machines is now often the most challenging aspect of their synthesis, as it was in the case of 18 . Therefore, one of the key challenges when simplifying the synthesis of prototype interlocked molecular machines is simplifying the production of requisite, often functionalized macrocycles.

At the other end of the complexity scale are interlocked molecular shuttles. Indeed, such switches make up the vast majority of interlocked molecular machines reported to date, and a very large range of behaviors and properties have been disclosed. (8) Given their relative structural simplicity compared with more advanced machines such asand, and longer history, it is perhaps unsurprising that their synthesis is extremely well developed, particularly the mechanical bond forming step, and thus, it might be expected that simple interlocked molecular switches are closer to real-world applications.

“Molecular synthesizers” 11 and 18 are extreme examples in terms of structural complexity and size, both of which are required to generate a complex outcome. The improvement in synthetic efficiency between generations is impressive and an example of the synthetic proficiency of many in the field that is not always widely recognized.

To overcome this, and in order to allow the general principle to be studied and expanded upon, the synthesis of subsequent generations of this machine was simplified dramatically. Specifically, whereas in the first-generation device the sequence information was included in the axle by laborious iterative synthesis, third-generation deviceuses a controlled radical polymerization process to generate functionalized axlewhich is then joined to preformed rotaxane fragment 2 Figure ). (19) Thus, although third-generation rotaxaneis synthesized in 10 linear steps, most of this effort relates to the synthesis of the macrocycle bearing the catalyst (7 steps), and the machine itself is produced in ∼10% yield with a total of 15 synthetic operations. Moreover, the operation of the machine results in the formation of an average of 6 new amide bonds, with 5% overall yield of the oligoamide product, a significant advance over first generation machine. The oligovaline product’s molecular weight and dispersity are determined by the functionalized polymeric axle fragment. The authors then demonstrated that the oligovaline product was a competent enantioselective catalyst, thus extending their proof-of-principle “artificial ribosome” to one capable of producing a functional product, in direct analogy with the equivalent natural molecular machine.

This suggests there is a need to balance blue-skies, proof-of-principle work with subsequent optimization and simplification of structure and synthesis. The progression of Leigh’s peptide synthesizing molecular machines (17) through various iterations is an excellent example of the first steps of this process. In 2013, Leigh and co-workers demonstrated that [2]rotaxane, in which the macrocycle bears a catalytic thiolate moiety, and the axle bears reactive phenolate esters, is able to “read” sequence information in the axle and “translate” this information into a chemical output in the form of a tripeptide with excellent sequence control ( Figure 1 ). (18) This groundbreaking result is clearly an example of proof-of-principle—the machine itself is far from the most efficient way of making a simple tripeptide! Indeed, although the synthesis ofwas designed to be highly convergent, requiring only 10 linear steps, the yield over this shortest sequence is <1%, and the whole route requires >25 synthetic operations. Thus, the overall yield of the product peptide sequence is ∼0.1% compared with ∼50% over 4 linear steps using solution phase techniques.

In contrast, in the synthesis of interlocked molecular machines, molecular complexity and the consequent synthetic challenge are a real issue; many advanced interlocked molecular machines are extremely challenging to access, and thus, vanishingly small quantities of material are typically available for study. This is despite the impressive progress in methodologies for forming the mechanical bond (9,15) since the first reports of synthetic rotaxanes and catenanes in the 1960s. (16) In the move from prototype to genuine application, this will matter more and more as the cost/benefit relationship of a new machine will largely relate to its cost of synthesis. Furthermore, because publications in the area focus, understandably, on the properties of the final machine, synthesis is often relegated to a brief comment (and a large electronic Supporting Information) despite representing a very large proportion of the project effort.

Examination of natural protein-based molecular machines reveals extremely large molecules in which the active domain(s) often represent a relatively small fraction of the molecular mass, with much of the rest of the machine playing a structural role by protecting active sites, anchoring the complex into position, assisting in transduction of molecular motion, etc. This appears to be a luxury that nature can afford; despite the inherent cost of such large structures, as they are produced efficiently, typically using other molecular machines that have evolved for the task, the benefit of the final function renders their synthesis a sound investment.

Simplicity of Operation ARTICLE SECTIONS Jump To

The operation of many interlocked molecular switches and motors is achieved chemically by iterative addition of reagents, for instance, acid followed by base. Although this is a very effective strategy for the investigation of new systems and behaviors, it clearly presents problems in terms of waste generation over multiple cycles in the context of applications. It also requires continuous significant intervention by the operator. Thus, despite the dominance of stimuli based on the addition of reagents, photochemical and electrochemical methods seem ideal for controlling the operation of interlocked molecular machines.

2. The electrochemical strategy introduced by Stoddart and co-workers in their original molecular shuttles remains one of the simplest available and led to one of the first proposed applications of molecular switches to catch the imagination of the field. In 2001, Stoddart, Heath, and co-workers created tunnel junction devices in which a monolayer of molecular shuttles was trapped between the cross bars. (33) Applying a write voltage switched the shuttles between the two available stations, resulting in a change in the resistance of the tunnel junction which could be read using a read voltage. In this way, Stoddart, Heath, and co-workers demonstrated the use of interlocked molecules in a “molecular memory” device with densities of up to 1011 bits/cm

38 begins by the electrochemical formation of a threaded pimer-complex between viologen macrocycle 39 and a viologen station in the “pump” region of the axle to give complex 40. This attractive interaction can then be abolished electrochemically simply by reoxidizing both macrocycle and axle to the closed-shell pyridinium forms. As this oxidation takes place, the positively charged macrocycle can either move away from the viologen binding site toward the pyridinium end of the axle, and ultimately freedom in solution, or move toward the neutral aromatic moiety. By optimizing the spacers between the viologen unit in the axle, and the pyridinium and aromatic blocking units, Stoddart and co-workers were able to kinetically bias this motion toward the neutral aromatic unit to give threaded species 41, presumably due to charge–charge repulsion between the cationic macrocycle and the cationic pyridinium moiety. Once it has gone the “wrong way”, the macrocycle becomes trapped as the dicationic viologen unit blocks its path electrostatically. The cycle completes via a slow, thermally activated slippage of the macrocycle over the neutral aromatic “speed bump” to give 42. More recently Stoddart and co-workers have demonstrated that this same simple approach can be used to control a much more complex molecular machine that they have designated a molecular pump ( Figure 4 ). (34) The operation ofbegins by the electrochemical formation of a threaded pimer-complex between viologen macrocycleand a viologen station in the “pump” region of the axle to give complex. This attractive interaction can then be abolished electrochemically simply by reoxidizing both macrocycle and axle to the closed-shell pyridinium forms. As this oxidation takes place, the positively charged macrocycle can either move away from the viologen binding site toward the pyridinium end of the axle, and ultimately freedom in solution, or move toward the neutral aromatic moiety. By optimizing the spacers between the viologen unit in the axle, and the pyridinium and aromatic blocking units, Stoddart and co-workers were able to kinetically bias this motion toward the neutral aromatic unit to give threaded species, presumably due to charge–charge repulsion between the cationic macrocycle and the cationic pyridinium moiety. Once it has gone the “wrong way”, the macrocycle becomes trapped as the dicationic viologen unit blocks its path electrostatically. The cycle completes via a slow, thermally activated slippage of the macrocycle over the neutral aromatic “speed bump” to give

Figure 4 Figure 4. Operation of Stoddart’s molecular pump, 38.

38, one macrocycle is pumped from solution onto a region of the axle with which it has no significant favorable interactions, and thus, a portion of the electrochemical energy inputted is stored. Furthermore, repeating a further complete cycle allows additional rings to be pumped onto the ring collecting portion of the axle to give 43. Later versions of this machine improved the efficiency of the system and allowed the synthesis of a [5]rotaxane containing four pumped rings. Overall, during one complete cycle of molecular machine, one macrocycle is pumped from solution onto a region of the axle with which it has no significant favorable interactions, and thus, a portion of the electrochemical energy inputted is stored. Furthermore, repeating a further complete cycle allows additional rings to be pumped onto the ring collecting portion of the axle to give. Later versions of this machine improved the efficiency of the system and allowed the synthesis of a [5]rotaxane containing four pumped rings. (35) This simple operational cycle suggests that such a machine could be used synthetically to generate unusual threaded polymers in which there is no interaction between the axle and ring, and even, as in this case, repulsive interactions between the rings themselves. Furthermore, embedding such a machine in a membrane would allow rings, and perhaps ultimately rings bearing a cargo, to be pumped against a concentration gradient using the same approach.

Light represents one of the simplest stimuli that can be applied to operate a molecular machine. (36) Typically, in the context of molecular switches, this requires the application of more than one wavelength of light: one to switch in one direction, the other to reverse the effect of the first. (37) Leigh and co-workers’ extensive use of fumaramide/maleamide isomerization in the development of molecular switches is an example of this approach, (38) as are shuttles based on azobenzene and stilbene units. (39)

44, that operates continuously and autonomously under irradiation with a single wavelength of light through a photoelectron transfer process (45 in which the affinity of the macrocycle for this station is reduced, and the system relaxes toward its new equilibrium position, 46, in which the macrocycle occupies the unsubstituted bipyridinium. Spontaneous back electron transfer regenerates the original stations (47), and the macrocycle then returns to its original equilibrium position (44). Thus, over one cycle of photon absorption, electron transfer, and back electron transfer the macrocycle undergoes net displacement from one station to the other and back again. In some cases, as in the case of shuttles based on the isomerization of azobenzene units, spontaneous thermal reversion to the initial state can allow the switching process to reverse without the application of a second stimulus. (39d) Taking this concept to its logical conclusion, but using a different mechanism, Balzani, Credi, Stoddart, and co-workers demonstrated a molecular shuttle,, that operates continuously and autonomously under irradiation with a single wavelength of light through a photoelectron transfer process ( Figure 5 a). (40) Upon absorption of the photon, electron transfer from the ruthenium-based stopper to the distal bipyridinium unit givesin which the affinity of the macrocycle for this station is reduced, and the system relaxes toward its new equilibrium position,, in which the macrocycle occupies the unsubstituted bipyridinium. Spontaneous back electron transfer regenerates the original stations (), and the macrocycle then returns to its original equilibrium position (). Thus, over one cycle of photon absorption, electron transfer, and back electron transfer the macrocycle undergoes net displacement from one station to the other and back again.

Figure 5 Figure 5. (a) Balzani, Credi, and Stoddart’s autonomous light-driven shuttle, 44. (b) Leigh’s photochemical information ratchet, 48. (c) Effect of an external photosensitizer on the distribution between the two compartments at photostationary state for 48.

44 is a molecular switch that spontaneously switches back and forth continuously under irradiation. Leigh and co-workers demonstrated a light-driven information ratchet 48 that operates under continuous irradiation with a single wavelength of light (48 operates due to the presence of two photosensitizers, one attached to the macrocycle and one exogenous sensitizer in solution. Irradiation leads to two sets of competing processes, the opening and closing of the α-methyl stilbene gate by the intramolecular sensitizer, which depends on the position of the macrocycle on the axle, and the opening and closing of the gate by the exogenous sensitizer, benzil, which does not. The interplay of these two processes leads to the macrocycle adopting a nonequilibrium distribution between the two compartments when the stilbene gate is closed (48 and (Z)-49, an effect that is enhanced in the presence of increasing equivalents of benzil. Rotaxaneis a molecular switch that spontaneously switches back and forth continuously under irradiation. Leigh and co-workers demonstrated a light-driven information ratchetthat operates under continuous irradiation with a single wavelength of light ( Figure 5 b). (41) Ratchetoperates due to the presence of two photosensitizers, one attached to the macrocycle and one exogenous sensitizer in solution. Irradiation leads to two sets of competing processes, the opening and closing of the α-methyl stilbene gate by the intramolecular sensitizer, which depends on the position of the macrocycle on the axle, and the opening and closing of the gate by the exogenous sensitizer, benzil, which does not. The interplay of these two processes leads to the macrocycle adopting a nonequilibrium distribution between the two compartments when the stilbene gate is closed ( Figure 5 c). When no benzil is present, irradiation leads to an equilibrium distribution between the two stations at steady state. Addition of benzil leads to a nonequilibrium distribution between (Z)-and ()-, an effect that is enhanced in the presence of increasing equivalents of benzil.

An alternative to using simple stimuli such as electro- and photochemistry is to develop systems that can operate autonomously without external intervention; indeed, most natural molecular machines take this approach. Leigh’s synthesizing molecular machines ( Figures 1 and 2 ) are artificial examples in that, once they are triggered, they will carry out their function without user intervention. Developing autonomous interlocked molecular motors capable of operating repetitively is challenging though, particularly those employing multiple steps that must be sequenced in time such as energy ratchet-based motors.

50 ( Recently, di Stefano and co-workers demonstrated the autonomous operation of rotaxane-based molecular switches using acidic reagents that slowly decompose to basic species, allowing pH-driven shuttles to switch through a complete cycle with a single user input. (42) This concept was subsequently elaborated into an autonomous molecular motor by Leigh and co-workers by combining acid/base switched stations with acid/base labilized blocking groups, ensuring the synchronization of the key chemical steps. (43) Catenane 6 Figure a) is an example of an energy ratchet-based motor in which the controlled protonation of the amine to give an ammonium unit with higher affinity for the macrocycle is synchronized with removal/reintroduction of the hydrazone and disulfide gates to generate a 360° rotation.

At high pH in the presence of NEt 3 , the amine station remains unprotonated, and the macrocycle preferentially occupies the triazolium station. Under these conditions the disulfide “gate” is under dynamic exchange between open and closed states through disulfide exchange. Addition of trichloroacetic acid (TCA) results in protonation of the amine station to generate a higher-affinity ammonium binding site for the macrocycle. The drop in pH shuts off the disulfide exchange reaction and initiates the dynamic exchange of the hydrazone gate. Thus, after addition of TCA the macrocycle can escape via the aldehyde branch of the large ring from the triazolium station to its new preferred ammonium binding site.

2 and CDCl 3 , the pH rises until the hydrazone exchange stops, and the disulfide exchange recommences. As the pH rises, the ammonium unit is deprotonated, and the preferred binding site is now the triazolium station. The macrocycle escapes to its new preferred equilibrium position via the thiol branch of the large macrocycle. Thus, overall, the motor undergoes a 360° rotation with each addition of the TCA “fuel” 2 and CDCl 3 the only waste products, allowing the cycle to be repeated with little or no fatigue. As the TCA decomposes to produce COand CDCl, the pH rises until the hydrazone exchange stops, and the disulfide exchange recommences. As the pH rises, the ammonium unit is deprotonated, and the preferred binding site is now the triazolium station. The macrocycle escapes to its new preferred equilibrium position via the thiol branch of the large macrocycle. Thus, overall, the motor undergoes a 360° rotation with each addition of the TCA “fuel” (44) with COand CDClthe only waste products, allowing the cycle to be repeated with little or no fatigue.

The motor behavior of catenane 50 is predicated on an energy ratchet mechanism that inherently relies on switching between two preferred equilibrium positions and the selective ungating/gating of the two different paths for the ring to escape to its new equilibrium position. The elegance of the operation mechanism developed is that all these steps are achieved by a simple change in pH that synchronizes both pairs of events. However, user intervention is still required at the end of each cycle, the addition of further TCA, to begin the next rotation.

Figure 6 Figure 6. (a) Autonomous catenane motor 50 that undergoes a single revolution upon addition of trichloroacetic acid. (b) Leigh’s autonomous information ratchet catenane motor that turns continuously in the presence of Fmoc-Cl.

52 contain near-identical fumaramide binding sites for the macrocycle (one station is deuterated for analytical purposes; however, the following discussion holds true even if both stations are identical). If one of the two gates is removed at random by a base-mediated elimination process, the macrocycle is free to explore both compartments and adopts an approximately 50:50 distribution between the near-isoenergetic stations. Reinstallation of the Fmoc group catalyzed by a bulky catalyst leads to kinetic discrimination in the unlinking reaction, which occurs most rapidly when the macrocycle occupies the station furthest from the alcohol functionality. In this way the small macrocycle undergoes a net half-rotation around the larger ring. Repeating this process while FmocCl remains, despite having no control over which Fmoc group is cleaved, results in continuous net rotation. Intriguingly this remains true despite the distribution of the macrocycle between the two compartments remaining at its equilibrium value of 50:50 at all times; one of the unusual features of machines like 52 is that they overcome the strictures of detailed balance, allowing continuous rotation of the motor even at steady state. Alternatively, information ratchet mechanisms can, in theory, operate autonomously and continuously if two reactions, the gating and ungating processes, can be designed to take place simultaneously, without any need to modify the affinity of the macrocycle for either compartment, by using kinetic factors to discriminate between the available pathways. Taking this approach, Leigh and co-workers designed a catenane information motor that undergoes continuous net rotation as long as a high-energy “fuel” (44) remains ( Figure 6 b). (45) The two compartments of catenanecontain near-identical fumaramide binding sites for the macrocycle (one station is deuterated for analytical purposes; however, the following discussion holds true even if both stations are identical). If one of the two gates is removed at random by a base-mediated elimination process, the macrocycle is free to explore both compartments and adopts an approximately 50:50 distribution between the near-isoenergetic stations. Reinstallation of the Fmoc group catalyzed by a bulky catalyst leads to kinetic discrimination in the unlinking reaction, which occurs most rapidly when the macrocycle occupies the station furthest from the alcohol functionality. In this way the small macrocycle undergoes a net half-rotation around the larger ring. Repeating this process while FmocCl remains, despite having no control over which Fmoc group is cleaved, results in continuous net rotation. Intriguingly this remains true despite the distribution of the macrocycle between the two compartments remaining at its equilibrium value of 50:50 at all times; one of the unusual features of machines likeis that they overcome the strictures of detailed balance, allowing continuous rotation of the motor even at steady state.