Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.
J Am Chem Soc
It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulﬁde bond reduction, a bimolecular nucleophilic substitution (SN2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulﬁde bond and obtain force- dependent rate constants of the disulﬁde bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (∆x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 ( 0.05 and 0.44 ( 0.03 Å for thiol-initiated and phosphine-initiated disulﬁde bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulﬁde bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the ∆x value for the phosphine-initiated reduction is decreased to 0.28 ( 0.04 Å whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an SN2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-ångström changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.