Search

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.

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.

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.

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.