Mechanical Activation Drastically Accelerates Amide Bond Hydrolysis, Matching Enzyme Activity.

Amide bonds, which include peptide bonds connecting amino acids in proteins and polypeptides, give proteins and synthetic polyamides their enormous strength. Although proteins and polyamides sustain mechanical force in nature and technology, how forces affect amide and peptide bond stability is still unknown. Using single-molecule force spectroscopy, we discover that forces of only a few hundred pN accelerate amide hydrolysis 109 -fold, an acceleration hitherto only known from proteolytic enzymes. The drastic acceleration at low force precedes a moderate additional acceleration at nN forces. Quantum mechanochemical ab initio calculations explain these experimental results mechanistically and kinetically. Our findings reveal that, in contrast to previous belief, amide stability is strongly force dependent. These calculations provide a fundamental understanding of the role of mechanical activation in amide hydrolysis and point the way to potential applications from the recycling of macromolecular waste to the design of bioengineered proteolytic enzymes.

Mechanochemical Cycloreversion of Cyclobutane Observed at the Single Molecule Level.

Mechanochemical cycloreversion of cyclobutane is known from ultrasound experiments. It is, however, not clear which forces are required to induce the cycloreversion. In atomic force microscopy (AFM) experiments, on the other hand, it is notoriously difficult to assign the ruptured bond. We have solved this problem through the synthesis of tailored macrocycles, in which the cyclobutane mechanophore is bypassed by an ethylene glycol chain of specific length. This macrocycle is covalently anchored between a glass substrate and an AFM cantilever by polyethylene glycol linkers. Upon mechanical stretching of the macrocycle, cycloreversion occurs, which is identified by a defined length increase of the stretched polymer. The measured length change agrees with the value calculated with the external force explicitly included (EFEI) method. By using two different lengths for the ethylene glycol safety line, the assignment becomes unambiguous. Mechanochemical cycloreversion of cyclobutane is observed at forces above 1.7 nN.

Mechanically induced silyl ester cleavage under acidic conditions investigated by AFM-based single-molecule force spectroscopy in the force-ramp mode.

AFM-based dynamic single-molecule force spectroscopy was used to stretch carboxymethylated amylose (CMA) polymers, which have been covalently tethered between a silanized glass substrate and a silanized AFM tip via acid-catalyzed ester condensation at pH 2.0. Rupture forces were measured as a function of temperature and force loading rate in the force-ramp mode. The data exhibit significant statistical scattering, which is fitted with a maximum likelihood estimation (MLE) algorithm. Bond rupture is described with a Morse potential based Arrhenius kinetics model. The fit yields a bond dissociation energy De = 35 kJ mol(-1) and an Arrhenius pre-factor A = 6.6 × 10(4) s(-1). The bond dissociation energy is consistent with previous experiments under identical conditions, where the force-clamp mode was employed. However, the bi-exponential decay kinetics, which the force-clamp results unambiguously revealed, are not evident in the force-ramp data. While it is possible to fit the force-ramp data with a bi-exponential model, the fit parameters differ from the force-clamp experiments. Overall, single-molecule force spectroscopy in the force-ramp mode yields data whose information content is more limited than force-clamp data. It may, however, still be necessary and advantageous to perform force-ramp experiments. The number of successful events is often higher in the force-ramp mode, and competing reaction pathways may make force-clamp experiments impossible.

A density functional theory model of mechanically activated silyl ester hydrolysis.

To elucidate the mechanism of the mechanically activated dissociation of chemical bonds between carboxymethylated amylose (CMA) and silane functionalized silicon dioxide, we have investigated the dissociation kinetics of the bonds connecting CMA to silicon oxide surfaces with density functional calculations including the effects of force, solvent polarizability, and pH. We have determined the activation energies, the pre-exponential factors, and the reaction rate constants of candidate reactions. The weakest bond was found to be the silyl ester bond between the silicon and the alkoxy oxygen atom. Under acidic conditions, spontaneous proton addition occurs close to the silyl ester such that neutral reactions become insignificant. Upon proton addition at the most favored position, the activation energy for bond hydrolysis becomes 31 kJ mol(-1), which agrees very well with experimental observation. Heterolytic bond scission in the protonated molecule has a much higher activation energy. The experimentally observed bi-exponential rupture kinetics can be explained by different side groups attached to the silicon atom of the silyl ester. The fact that different side groups lead to different dissociation kinetics provides an opportunity to deliberately modify and tune the kinetic parameters of mechanically activated bond dissociation of silyl esters.