Among the array of complex terpene-forming carbocation cyclization/rearrangement reactions, the so-called "triple shift" reactions are among the most unexpected. Such reactions involve the asynchronous combination of three 1,n-shifts into a concerted process, e.g., a 1,2-alkyl shift followed by a 1,3-hydride shift followed by a second 1,2-alkyl shift. This type of reaction so far has been proposed to occur during the biosynthesis of diterpenes and the sidechains of sterols. Here we describe efforts to push the limits of concertedness in this type of carbocation reaction by designing, and characterizing with quantum chemical computations, systems that could couple additional 1,n-shift events to a triple shift leading, in principle to quadruple, pentuple, etc. shifts. While our designs did not lead to clear-cut examples of quadruple, etc. shifts, they did lead to reactions with surprisingly flat energy surfaces where more than five chemical events connect reactants and plausible products. Ab initio molecular dynamics simulations demonstrate that the formal minima on these surfaces interchange on short timescales, both with each other and with additional unexpected structures, allowing us a glimpse into a very complex manifold that allows ready access to great structural diversity.

Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2'OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2'OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.