Force spectroscopy allows the measurement of reaction rates as a function of the restoring force in molecules that have been stretched or compressed, but it lacks the temporal and spatial resolution needed to study small functional groups. A molecular force probe that extends force spectroscopy to the size-scale of such reactions has now been reported by roman Boulatov and co-workers. This artist's impression shows how stiff stillbene molecules can be used to apply forces to a small functional group: the stiff stilbene molecule changes shape when it is exposed to certain wavelengths of light, and this changes the force experienced by the smaller molecule. Cover Page

Mechanochemistry: Tug of war Research Highlights Nature 458, 552 (2 April 2009) Mechanochemistry: Tug of war Even the strongest molecular bonds break if yanked hard enough. But studying this effect requires a delicate tugging mechanism that can focus force controllably on individual bonds. Roman Boulatov and his colleagues at the University of Illinois in Urbana-Champaign have found such a device: a rigid U-shaped molecule, stiff stilbene (pictured), the ends of which are attached to the molecule under interrogation. Stilbene twists into a strained shape on exposure to light, pulling on its attached molecule. The force generated can be calculated from quantum mechanical principles, and altered incrementally depending on the length of an adjustable linker. The researchers confirm a direct relationship between the force their probe exerts on a cyclobutene molecule and the rate at which a central bond falls apart.

The capacity of living organisms to convert conformational changes in a reacting molecule into directional motion has stimulated considerable effort to understand the underlying principles and to realize them in synthetic systems. The simplest, paradigmatic example is a single molecule of E-oligoazobenzene stretched between an AFM tip and a glass slide. Its contraction on irradiation brings the tip closer to the slide by bending the AFM cantilever. Here the macromolecule is constrained along a single axis into a nonequilibrium configuration which creates a gradient of the molecular free energy (restoring force) along this axis. This restoring force is balanced by the elastic force of the bent cantilever. Irradiation-induced contraction of the oligomer along the constrained axis suggests an increase in the restoring force upon electronic excitation of azobenzene and is consistent with the current understanding of the topologies of the ground and excited-state surfaces of free azobenzene.

Here we show that a molecular force probe instead of a macroscopic one, such as AFM, can be used both to constrain a small molecule (substrate) along a single axis and to estimate the resultant restoring force. This molecular force probe is a small reactant whose thermally accessible transition state allows a partial relaxation of the constrained substrate primarily along a single axis (Figure 2).

A molecular force probe would be an inert molecule whose restoring force can be varied systematically in ,50 pN increments over a range of at least 500 pN by constraining a single internuclear distance. By incorporating a functional group of interest (substrate) in the linker that constrains this distance and varying the length and/or conformational flexibility of this linker, a homologous series of macrocycles with increasing restoring force is synthesized. Standard methods of chemical kinetics are suitable for measuring the activation enthalpies and entropies of the substrate reaction. The restoring forces are available from high-level quantumchemical calculations, for which accuracy is benchmarked against the experimental activation parameters. Studies of functional groups’ reactivities using microscopic force probes are complicated not only by the inherent challenges of resolving localized reactions in macromolecules, but also by the difficulties in measuring activation parameters, the lack of product characterization and the need for empirical models to estimate the molecular restoring force from distortions of the force probe, with the concomitant uncertainties in the relative orientations of the force vector and the reacting moiety

Figure 1 | Microscopic versus molecular force probes Figure 1 | Microscopic versus molecular force probes. Force spectroscopy enables the study of reaction kinetics as a function of the restoring force of a stretched or compressed reactant. Microscopic force probes (left panels) are suited for reactions involving nanometre-scale structural changes such as protein unfolding. However, their surface roughness and thermal fluctuations obscure most localized chemical reactions. Thermal fluctuations are amenable to study with a molecular force probe such as stiff stilbene (red moiety in the right panels), an inert molecular fragment with a restoring force that is easily incrementable over hundreds of piconewtons (pN) by constraining a single internuclear distance. Series of strained macrocycles containing E stiff stilbene are distinct from all previously reported strained molecules in allowing the application of the restoring force formalism, which has hitherto been limited to reactions of polymers, to much smaller and more tractable molecules. The linkers are connected to the C6,C60 atoms of stiff stilbene.

The large difference in the C6 The large difference in the C6...C6` distance between the Z and E isomers, the high thermal barrier that separates the isomers and their clean photoisomerization by 400 nm light makes macrocycles with up to 30 kcal mol-1 of strain energy readily available upon irradiation of strain-free Z analogues. The availability of strain-free Z isomers offers an additional advantage in that the differences in the activation parameters between the two isomers can be measured and calculated with higher accuracy than the individual values and are free from kinetic perturbations such as polar effects, which are not accommodated within the force formalism.

Figure 3 | Kinetic and force data for macrocycles 1–9 Figure 3 | Kinetic and force data for macrocycles 1–9. a, Measured (blue) and calculated (red) difference in the activation enthalpies DDH‡ of the substrate reaction in the Z and E isomers of the same macrocycle. The error bars define the 95% confidence interval. The activation entropies were small and varied little across the series (see Supplementary Table S9). b, Restoring forces of the non-reactive part of the macrocycle were calculated quantumchemically using fragments obtained by replacing C4H4 with a pair of hydrogen atoms (example of E–1 is shown, carbon, oxygen and hydrogen atoms are grey, red and yellow, respectively). The forces are presented as components along and orthogonal to the CH3...CH3 axis; signifies forces averaged over multiple structures along the reaction path. For all fragments the total restoring force of one indanyl group and its attached linker was equal in magnitude and opposite in sign to that of the other indanyl group and its linker, regardless of the fragment symmetry.