Rearrangement-Free Hydroxylation of Methylcubanes by a Cytochrome P450: The Case for Dynamical Coupling of C-H Abstraction and Rebound.

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The highly strained cubylmethyl radical undergoes one of the fastest radical rearrangements known (reported k = 2.9 × 1010 s-1 at 25 °C) through scission of two bonds of the cube. The rearrangement has previously been used as a mechanistic probe to detect radical-based pathways in enzyme-catalyzed C-H oxidations. This paper reports the discovery of highly selective cytochrome P450-catalyzed methylcubane oxidations which notionally proceed via cubylmethyl radical intermediates yet are remarkably free of rearrangement. The bacterial cytochrome P450 CYP101B1 from Novosphingobium aromaticivorans DSM 12444 is found to hydroxylate the methyl group of a range of methylcubane substrates containing a regio-directing carbonyl functionality at C-4. Unlike other reported P450-catalyzed methylcubane oxidations, the designed methylcubanes are hydroxylated with high efficiency and selectivity, giving cubylmethanols in yields of up to 93%. The lack of cubane core ring-opening implies that the cubylmethyl radicals formed during these CYP101B1-catalyzed hydroxylations must have very short lifetimes, of just a few picoseconds, which are too short for them to manifest the side reactivity characteristic of a fully equilibrated P450 intermediate. We propose that the apparent ultrafast radical rebound can be explained by a mechanism in which C-H abstraction and C-O bond formation are merged into a dynamically coupled process, effectively bypassing a discrete radical intermediate. Related dynamical phenomena can be proposed to predict how P450s may achieve various other modes of reactivity by controlling the formation and fate of radical intermediates. In principle, dynamical ideas and two-state reactivity are each individually able to explain apparent ultrashort radical lifetimes in P450 catalysis, but they are best considered together.