Substituted Norcaranes as Mechanistic Radical Clock Probes: Characterization of Radical Rearrangement

Norcarane (bicyclo4.1.0heptane) derivatives are valuable mechanistic probes for distinguishing concerted, radical, and cation-mediated enzyme mechanisms. To investigate the role of enzyme-substrate complementarity in cytochrome P450 catalyzed reactions, ester-functionalized norcarane probes were previously designed for tight binding to the active sites of some of these enzymes. To evaluate the potential of the norcarane probes to detect radical intermediates, the pyridine-2-thione-N-oxycarbonyl (PTOC) esters of both endo- and exo-bicyclo4.1.0hept-2-yl butyrate were synthesized as radical precursors in 6-8 steps, respectively. Photolysis of the PTOC esters allowed measurement of radical rearrangement rates, with both the endo (1.6 ± 0.1 × 108 s-1) and exo (1.9 ± 0.2 × 108 s-1) systems giving values comparable to those reported for the parent norcarane scaffold (2 × 108 s-1). The identity of these rearrangement products was confirmed through comparison with authentic synthetic standards. These results validate the herein reported functionalized norcaranes as radical clocks for studying P450 enzymes, as they have well-characterized, predictable, and reproducible radical rearrangements.