N-dealkylation of an N-cyclopropylamine by horseradish peroxidase. Fate ofthe cyclopropyl group

Cyclopropylamines inactivate cytochrome P450 enzymes which catalyze their o xidative N-dealkylation. A key intermediate in both processes is postulated to be a highly reactive aminium cation radical formed by single electron t ransfer (SET) oxidation of the nitrogen center, but direct evidence for thi s has remained elusive. To address this deficiency and identify the fate of the cyclopropyl group lost upon N-dealkylation, we have investigated the o xidation of N-cyclopropyl-N-methylaniline (3) by horseradish peroxidase, a well-known SET enzyme. For comparison, similar studies were carried out in parallel with N-isopropyl-N-methylaniline (9) and N,N-dimethylaniline (8). Under standard peroxidatic conditions (HRP, H2O2, air), HRP oxidizes 8 comp letely to N-methylaniline (4) plus formaldehyde within 15-30 nun, whereas 9 is oxidized more slowly (< 10% in 60 min) to produce only N-isopropylanili ne (10) and formaldehyde (acetone and 4 are not formed). In contrast to res ults with 9, oxidation of 3 is complete in < 60 min and affords 4 (20% yiel d) plus traces of aniline. By using [1 '-C-14]-3, [1 '-C-13]-3, and [2 ' ,3 '-C-13]-3 as substrates, radiochemical and NMR analyses of incubation mixt ures revealed that the complete oxidation of 3 by HRP yields 4 (0.2 mol), b eta -hydroxypropionic acid (17, 0.2 mol), and N-methylquinolinium (16, 0.8 mol). In buffer purged with pure O-2, the complete oxidation of 3 yields 4 (0.7 mol), 17 (0.7 mol), and 16 (0.3 mol), while under anaerobic conditions , 16 is formed quantitatively from 3. These results indicate that the amini um ion formed by SET oxidation of 3 undergoes cyclopropyl ring fragmentatio n exclusively to generate a distonic cation radical (14(+.)) which then par titions between unimolecular cyclization (leading, after further oxidation, to 16) and bimolecular reaction with dissolved oxygen (leading to 4 and 17 in a 1:1 ratio). Neither beta -hydroxypropionaldehyde, acrolein, nor cyclo propanone hydrate are formed as SET metabolites of 3. The synthetic and ana lytical methods developed in the course of these studies should facilitate the application of cyclopropylamine-containing probes to reactions catalyze d by cytochrome P450 enzymes.