Regiochemical Variations in Reactions of Methylcubane with tert-Butoxyl Radical, Cytochrome P-450 Enzymes, and a Methane Monooxygenase System

Reactions of methylcubane ( 1 ) with the tert -butoxyl radical ( t -BuO • ), with cytochrome P-450 enzymes, and with a methane monooxygenase (MMO) system have been studied. For the purpose of product characterization, authentic samples of 2-methylcubyl and 4-methylcubyl derivatives were prepared. 2-Methylcubanecarboxylic acid ( 9b ) is a new compound prepared from cubanecarboxylic acid. The key synthetic reactions were (1) metalation and subsequent iodination of the 2-position of (diisopropylcarbamoyl)cubane to effect the initial functionalization, (2) lithium-for-iodine exchange and methylation followed by reduction to give 2-methyl-1-[(diisopropylamino)methyl]cubane, and (3) dimethyldioxirane oxidation of this amine to give 9b . The known 4-methylcubanecarboxylic acid ( 9d ) was prepared here by a route related to that employed for 9b . Reactions of acids 9b and 9d with methyllithium gave the corresponding methyl ketones which were oxidized by m -chloroperoxybenzoic acid to provide authentic samples of 2- and 4-methylcubanol acetates ( 3b and 3d ). Reaction of 1 with t -BuO • in the presence of 2,2,5,5-tetramethylisoindole- N -oxyl radical (TMIO • ) at 40−55 °C gave mainly cube-substituted products in confirmation of the report (Della, E. W.; Head, N. J.; Mallon, P.; Walton, J. C. J. Am. Chem. Soc. 1992, 114, 10730) that hydrogen atom abstraction by the electrophilic alkoxyl radical at low temperature occurs at the cubyl C−H positions. In a competition experiment at 42 °C, methylcubane was at least 3.5 times more reactive toward t -BuO • than cyclohexane, indicating that the cubyl positions in 1 are ≥40 times more reactive than the methyl positions in 1 (per hydrogen) toward the alkoxyl radical. Oxidation of 1 by enzymes gave alcohol products that were converted to their acetate derivatives for identification and quantitation. Microsomal cytochrome P-450 enzymes from rat and the rat purified P-450 isozyme CYP2B1 hydroxylated 1 at all positions, whereas the reconstituted MMO system from Methylococcus capsulatus (Bath) hydroxylated 1 only at the methyl position. The differences in regioselectivity suggest that the transition states for hydrogen abstraction by the alkoxyl radical and for enzyme-catalyzed hydroxylation differ considerably. The results are consistent with a model for concerted enzyme catalyzed hydroxylation of 1 involving “side-on” approach to the C−H bond of substrate.