Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo [4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J., accompanying article). In the present work, the product profiles from oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2-D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPΔ2E1, CYPΔ2E1 T303A, and CYPΔ2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn-and anti-2-norcarene oxide, syn-and anti-3-norcarene oxide, syn-and anti-4-hydroxy-2-norcarene, syn-and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5 to 15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations.Mechanistic probes have been used for years to reveal details about reaction mechanisms in chemistry and biology. The concept of a mechanistic probe study is that a short-lived intermediate can be revealed by a characteristic rearrangement of a probe substrate that is observed in the reaction products. One compound used as a probe substrate for studies of enzyme-catalyzed oxidations is norcarane, bicyclo[4.1.0]heptane (1). During the course of probe studies with norcarane in our laboratories, 1 we observed a large number of minor oxidation products that appeared to be derived from norcarane as judged by GC retention times and mass spectra. Eventually, we realized that, in addition to expected alcohol products, the enzymes were oxidizing norcarane to both 2-norcarene (2) and 3-norcarene (3), and that these alkenes were further oxidized to give small amounts of secondary oxidation products. of the norcarane oxidation studies are reported in the accompanying paper. 3 In this paper, we report studies of the oxidations of both 2-norcarene and 3-norcarene by several iron-containing enzymes. Most of the products from oxidations of th...

Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPΔ2B4, CYPΔ2E1, and CYPΔ2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radicalderived product from oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and putative cationderived product from oxidation of norcarane, cyclohept-3-enol (22), co-elute with other oxidation products on low polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and, therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than previously reported.Mechanistic probes have been used for many years to reveal details about reaction mechanisms in chemistry and biology. The concept of a mechanistic probe study is that a short-lived intermediate can be revealed by a characteristic rearrangement of a probe substrate that is observed in the reaction products. Because transient species are not followed in real time, the existence of an intermediate can only be inferred, and the validity of conclusions based on the use of probes depends on a thorough understanding of potentially complex chemistry. Norcarane (1) has been applied as a probe in studies of many enzyme-catalyzed oxidation reactions. 1-10 In most of these works, small amounts of a putative radical-derived product and a putative cation-derived product were detected, providing evidence that radicals and cations were formed as intermediates to some extent in the oxidation reactions. The evidence for radical-derived products in norcarane studies and the conclusions that radical intermediates existed is essentially unique. Most probes that were used in studies of oxidizing enzymes give the same skeletal rearrangement from radical and cation intermediates, and one cannot conclude what type of transient was involved when rearranged products are found. The ev...