Dynamics of individual molecular shuttles under mechanical force

Molecular shuttles are the basis of some of the most advanced synthetic molecular machines. In these devices a macrocycle threaded onto a linear component shuttles between different portions of the thread in response to external stimuli. Here, we use optical tweezers to measure the mechanics and dynamics of individual molecular shuttles in aqueous conditions. Using DNA as a handle and as a single molecule reporter, we measure thousands of individual shuttling events and determine the force-dependent kinetic rates of the macrocycle motion and the main parameters governing the energy landscape of the system. Our findings could open avenues for the real-time characterization of synthetic devices at the single molecule level, and provide crucial information for designing molecular machinery able to operate under physiological conditions.


S1.1 Chemical synthesis of the molecular shuttle
The chemical structure of the shuttle comprises a strong binding station, namely fumaramide (green in Fig. 1a), which is known to be the best fit for a tetraamide macrocycle, because its two amide carbonyls are held by the trans double bond in the correct geometry to form four strong hydrogen bonds with the macrocycle. Separated by an oligoethyleneglycol spacer of approximately 15 nm in length (see below) we placed a succinic amide-ester station, (orange in Fig. 1a). This station shows less affinity for the macrocycle than the fumaramide station, due to its flexibility and the substitution of one of the amides with an ester, which is a significantly weaker H-bond acceptor. At the ends of the polar spacer two bulky diphenylethyl groups serve as stoppers to prevent unthreading of the macrocycle. One of the stopper ends is branched to include a biotin group. The synthesis of the axle for the shuttle was carried out in 12 steps (see below). Macrocyclization around the fumaramide station was performed using a purposely synthesized U-shape (compound 14) The macrocycle contains an azide-decorated isophthalic acid chloride derivative (compound 13) at a single position for the covalent attachment of a polydT oligonucleotide.

(Z/E 55/45)-Thread
Compound 6 (85 mg, 0.13 mmol) was dissolved in DCM (3 mL) and the solution was cooled to 0ºC, EDCI (23 mg, 0.19 mmol), DMAP (37 mg, 0.19 mmol) were added at 0ºC. The reaction mixture was allowed to stir at room temperature for 30 min, and then a solution of compound 10 (180 mg, 0.12 mmol) in DCM (7.7 mL) was added to the activated acid. The reaction mixture was stirred overnight, concentrated under reduced pressure and then diluted with DCM. The organic layer was washed with 1M HCl, with NaHCO 3 (sat. aq.), then further washed with brine (sat. aq.), dried over Na 2 SO 4 and concentrated under reduced pressure. The crude material was purified by column chromatography (gradient elution: DCM/MeOH 30:1 to 9:1) to furnish the desired product as a colorless oil, 166mg, 0.077 mmol (64%

5-azidoisophthaloyl dichloride
Step i) 5-aminoisophalic acid (2.5 g, 27.6 mmol) was placed in a flash with water (39 mL), 3 mL of 12M HCl was added dropwise. The mixture was cooled in a ice bath at 0ºC. NaNO 2 was dissolved in water and added dropwise to the mixture, which was stirred for 30 min. Then, NaN 3 dissolved in water was added dropwise to the mixture. A yellow solid was formed and gas evolution was observed, making it difficult to keep stirring. The mixture was stirred until gas evolution was no longer detected. The product was then filtered, washed with distilled water and dried under vacuum (3.4g, 60% Step ii) To a stirred suspension of 5-azido-isophthalic acid (100 mg, 0.48 mmol) in DCM (2 mL) two drops of anhydrous DMF and oxalyl chloride (0.15 mL, 1.95 mmol) were added. The reaction mixture was stirred until the product was totally solubilized. The solvent was removed under reduced pressure and the sample was kept 3 h under vacuum to remove oxalyl chloride. The crude product was then used directly in the next reaction step.
Step iii) Compound 18 was dissolved in DCM, the solution was cooled to 0 ºC and TFA was added dropwise until the reaction was completed (TLC). Solvent was removed under vacuum.
The crude material was dissolved in DCM/MeOH 1/1 v/v ratio and was stirred with Amberlyst