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We report on the use of atomic force microscopy (AFM) to identify and characterize an intermediate state in macrocycle shuttling in a hydrogen bonded amide-based molecular shuttle. The [2]rotaxane consists of a benzylic amide macrocycle mechanically locked onto a thread that bears both fumaramide and succinic amide-ester sites, each of which can bind to the macrocycle through up to four intercomponent hydrogen bonds. Using AFM-based single-molecule force spectroscopy, we mechanically triggered the translocation of the ring between the two principal binding sites ("stations") on the axle. Equilibrium fluctuations reveal another interacting site involving the two oxygen atoms in the middle of the thread. We characterized the ring occupancy distribution over time, which confirms the intermediate in both shuttling directions. The study provides evidence of weak hydrogen bonds that are difficult to detect using other methods and shows how the composition of the thread can significantly influence the shuttling dynamics by slowing down the ring motion between the principal binding sites. More generally, the study illustrates the utility that single-molecule experiments, such as force spectroscopy, can offer for elucidating the structure and dynamics of synthetic molecular machines.

Donor-acceptor (DA) π-interactions are weak attractive forces that are exploited widely in molecular and supramolecular chemistry. They have been characterized extensively by ensemble techniques, providing values for their energies that are useful for the design of soft materials. For implementation of motions or operations based on these DA π-interactions in wholly synthetic molecular machines, the mechanical strength and force associated with their out-of-equilibrium performance are the key parameters, in addition to their energies obtained at thermodynamic equilibrium. In this context, we have used single-molecule force spectroscopy as a nonequilibrium technique to determine the mechanical strength of individual DA π-interactions in solution. We designed and synthesized a molecular tweezer that is able to encapsulate π-donors and also demonstrated a precise opening extension. The mechanical breaking of the noncovalent interactions between viologen units-π-acceptors commonly employed in mechanically interlocked molecules-and several π-donors afforded a characteristic force-distance signature, revealing the opening of individual viologen tweezers with an unambiguous extension. Single-tweezer host-exchange experiments performed

Secondary a-helix and b-sheet structures are key scaffolds around which the rest of the residues condense during protein folding. They are crucial for proteins to adopt their correct native structure. Despite their key role in numerous processes to maintain life, little is known about their properties under force. Their stability under mechanical stress, as constantly experienced in the turbulent environment of cells, is however essential. Here, we designed and synthesized two pH-responsive polypeptides, poly(L-glutamic acid) and poly(L-lysine), for optimal interfacing with an AFM single-molecule force spectroscopy set-up to probe the mechanical unfolding of a-helix and b-sheet secondary motifs. The force experiments, supported by simulations, reveal a superior mechanical stability of the poly(L-Lysine) a-helix, which we attribute to hydrophobic interactions of the alkyl side chains. Most importantly, our results show that these interactions play a key role in inhibiting the formation of a metastable b-sheet-like structure when the polypeptide is subjected to mechanical deformations, which might have important implications in the mechanism behind polyQ diseases.

Artificial molecular pumps (AMPs), inspired by the active cellular transport exhibited in biological systems, enable cargoes to undergo unidirectional motion, courtesy of molecular ratchet mechanisms in the presence of energy sources. Significant progress has been achieved, using alternatively radical interactions and Coulombic repulsive forces to create working AMPs. In an attempt to widen the range of these AMPs, we have explored the effect of molecular pumping on the photophysical properties of a collecting chain on a dumbbell incorporating a centrally located pyrene fluorophore and two terminal pumping cassettes. The AMP discussed here sequesters two tetracationic cyclophanes from the solution, generating a [3]rotaxane in which the fluorescence of the dumbbell is quenched. The research reported in this Article demonstrates that the use of pumping cassettes allows us to generate the [3]rotaxane in which the photophysical properties of fluorophores can be modified in a manner that cannot be achieved with a mixture of the dumbbell and ring components of the rotaxane on account of their weak binding in solution.

Mechanically interlocked molecules (MIMs), such as rotaxanes, catenanes, and molecular knots, have attracted significant interest because of their unique properties originating from their mechanically bonded components. Recently, MIMs have been employed in increasingly diverse architectures thanks to the tools of rational molecular design and the ability to incorporate functions in a precise manner. Here, we discuss advances in MIM synthesis, the fundamental understanding of their working processes, and applications exploiting their singular behavior. This review covers some of the most recent studies demonstrating the widespread interest in MIMs by scientists pursuing the ultimate goal of designing functional molecular machines that surpass their natural analogs or exhibit unprecedented properties.

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch‐based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non‐dissociative electron transfer as a reductive stimulus in the gas phase and probe the consecutive co‐conformational transitions of a donor‐acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. A comparison of collision cross section distributions for analogous closed‐shell and radical molecular ions sheds light on their respective formation energetics, while variations in their respective infrared absorption bands evidence changes in intramolecular organization as the foldamer becomes more compact. These differences are compatible with the advent of radical‐pairing interactions., Gas‐phase non‐dissociative electron transfer is used for charge reduction of a donor‐acceptor oligorotaxane foldamer (green). The consecutive co‐conformational transition is monitored using ion mobility spectrometry and infrared ion spectroscopy. Comparing the collision cross section distributions (blue) and infrared spectra (red) recorded for analogous closed‐shell and radical systems highlights differences that can be attributed to radical‐pairing interactions.

Mussel wet adhesion is known for its outstanding strength on a variety of surfaces. On the basis of the hypothesis that 3,4-dihydroxyphenylalanine, a catecholic amino acid, governs mussel adhesion, chemists have put much effort into the design of adhesive synthetic polymers containing catechols. However, the exceptional properties exhibited by the native proteins were hardly captured. The attempts to make those polymers stick to wet inorganic surfaces resulted in low adhesive forces. Here we synthesized poly(dopamine acrylamide) and measured the interaction forces with various inorganic surfaces using atomic force microscopy-based single-molecule force spectroscopy. We show that hydroxylation of the surface plays a pivotal role on the formation of strong bonds. We demonstrate that depending on the conditions, the whole range of interactions, from weak interactions to covalent bonds, can come into play.

International audience; α-Helix is the most predominant secondary structure in proteins and supports many functions in biological machineries. The conformation of the helix is dictated by many factors such as its primary sequence, intramolecular interactions, or the effect of the close environment. Several computational studies have proposed that there is a critical maximum length for the formation of intact compact helical structures, supporting the fact that most intact α-helices in proteins are constituted of a small number of amino acids. To obtain a detailed picture on the formation of α-helices in peptides and their mechanical stability, we have synthesized a long homopolypeptide of about 90 amino acids, poly(γ-benzyl-L-glutamate), and investigated its mechanical behaviour by AFM-based single-molecule force spectroscopy. The characteristic plateaus observed in the force–extension curves reveal the unfolding of a series of small helices (from 1 to 4) of about 20 amino acid residues connected to each other, rather than a long helix of 90 residues. Our results suggest the formation of a tertiary structure made of short helices with kinks, instead of an intact compact helical structure for sequences of more than 20 amino acid residues. To our knowledge, this is the first experimental evidence supporting the concept of a helical critical length previously proposed by several computational studies.

Over the past decades, chemists have been pursuing the creation of man-made molecular machines with either designed engineering-like operations or with higher performances compared with biological machines. The promise of creating an artificial molecular world traces its origins in the well-known lecture of Richard Feynman, There’s plenty of room at the bottom (1). Feynman’s insights into the immense possibilities of such small artificial machines, assembled in a straightforward manner, were deeply inspiring for the scientific community. The design of machines on the molecular scale is not an easy task to accomplish. Instead of gravity and inertia, which are omnipresent in the macroscopic world, random thermal fluctuations are prevalent and dominate movements on the molecular scale. Two main approaches are being considered for the construction of artificial molecular machines (AMMs): namely, bio-inspiration and miniaturization. The former consists of integrating concepts from naturally occurring machines and unnatural building blocks into AMMs, while the latter involves engineering nano-devices based on the mechanical actions of macroscopic machines. Perhaps a better way to design AMMs with unprecedented functions would be to follow neither of these routes, but rather to construct molecularly precise architectures based on recent advances in supramolecular chemistry: explore what has not been built by Nature. This unnatural route would only share with the biological world its fundamental laws at small scales, but differentiates it from its working processes. It is clear that the design of AMMs is a critical step in the process. It requires novel chemical building blocks to assemble and produce functioning systems. In this regard, mechanically interlocked molecules (MIMs) have paved the way for the synthesis of AMMs. MIMs, introduced by Jean-Pierre Sauvage (2) more than 30 years ago, signaled a breakthrough in introducing a new type of bond—the mechanical bond (3)—into chemistry. Examples of rotaxanes, catenanes, and other … [↵][1]1To whom correspondence should be addressed. Email: stoddart{at}northwestern.edu. [1]: #xref-corresp-1-1

Wholly synthetic molecules involving both mechanical bonds and a folded secondary structure are one of the most promising architectures for the design of functional molecular machines with unprecedented properties. Here, we report dynamic single-molecule force spectroscopy experiments that explore the energetic details of donor–acceptor oligorotaxane foldamers, a class of molecular switches. The mechanical breaking of the donor–acceptor interactions responsible for the folded structure shows a high constant rupture force over a broad range of loading rates, covering three orders of magnitude. In comparison with dynamic force spectroscopy performed during the past 20 y on various (bio)molecules, the near-equilibrium regime of oligorotaxanes persists at much higher loading rates, at which biomolecules have reached their kinetic regime, illustrating the very fast dynamics and remarkable rebinding capabilities of the intramolecular donor–acceptor interactions. We focused on one single interaction at a time and probed the stochastic rupture and rebinding paths. Using the Crooks fluctuation theorem, we measured the mechanical work produced during the breaking and rebinding to determine a free-energy difference, Δ G , of 6 kcal·mol −1 between the two local conformations around a single bond.

At the interface between foldamers and mechanically interlocked molecules, oligorotaxanes exhibit a spring-like folded secondary structure with remarkable mechanical and physicochemical properties. Among these properties, the ability of oligorotaxanes to act as molecular switches through controlled modulations of their spatial extension over (un)folding dynamics is of particular interest. The present study aims to assess and further characterize this remarkable feature in the gas phase using mass spectrometry tools. In this context, we focused on the [4]5NPR+12 oligorotaxane molecule complexed with PF6– counterion and probed its co-conformational states as a function of the in-source-generated charge states. Data were interpreted in light of electronic secondary structure computations at the PM6 and DFT levels. Our results highlight two major co-conformational groups associated either with folded compact structures, notably stabilized by intramolecular π–π interactions and predominant for low charge state...

Folding is a ubiquitous process that nature uses to control the conformations of its molecular machines, allowing them to perform chemical and mechanical tasks. Over the years, chemists have synthesized foldamers that adopt well-defined and stable folded architectures, mimicking the control expressed by natural systems 1,2 . Mechanically interlocked molecules, such as rotaxanes and catenanes, are prototypical molecular machines that enable the controlled movement and positioning of their component parts 3–5 . Recently, combining the exquisite complexity of these two classes of molecules, donor–acceptor oligorotaxane foldamers have been synthesized, in which interactions between the mechanically interlocked component parts dictate the single-molecule assembly into a folded secondary structure 6–8 . Here we report on the mechanochemical properties of these molecules. We use atomic force microscopy-based single-molecule force spectroscopy to mechanically unfold oligorotaxanes, made of oligomeric dumbbells incorporating 1,5-dioxynaphthalene units encircled by cyclobis(paraquat-p-phenylene) rings. Real-time capture of fluctuations between unfolded and folded states reveals that the molecules exert forces of up to 50 pN against a mechanical load of up to 150 pN, and displays transition times of less than 10 μs. While the folding is at least as fast as that observed in proteins, it is remarkably more robust, thanks to the mechanically interlocked structure. Our results show that synthetic oligorotaxanes have the potential to exceed the performance of natural folding proteins.

The force-driven separation of double-stranded DNA is crucial to the accomplishment of cellular processes like genome transactions. Ligands binding to short DNA sequences can have a local stabilizing or destabilizing effect and thus severely affect these processes. Although the design of ligands that bind to specific sequences is a field of intense research with promising biomedical applications, so far, their effect on the force-induced strand separation has remained elusive. Here, by means of AFM-based single molecule force spectroscopy, we show the co-existence of two different mechanisms for the separation of a short DNA duplex and demonstrate how they are perturbed by small binders. With the support of Molecular Dynamics simulations, we evidence that above a critical pulling rate one of the dissociation pathways becomes dominant, with a dramatic effect on the rupture forces. Around the critical threshold, we observe a drop of the most probable rupture forces for ligand-stabilized duplexes. Our results offer a deep understanding of how a stable DNA–ligand complex behaves under force-driven strand separation.

Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.