Products from enzyme-catalyzed oxidations of norcarenes

Abstract

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. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the 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 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, 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 1.5-15 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.

Figure 1

Selected coupling constants (top) and NOE enhancements (bottom) for epoxide 7. The NOE values are for irradiation at H_C1 and H_C2.

Figure 2

(A) GC retention times for compounds 4-16 on a low polarity column (5% phenyl silicone) at 80 °C. (B) Expansion of the congested region of the trace in A with the GC trace of products from oxidation of 3-norcarene by CYPΔ2E1 (gray line) superimposed. The compound numbers are shown above lines that indicate the retention times for each compound. Allylic alcohols 10 and 13 had the same retention times to within 0.01 min.

Figure 3

(A) Mass spectral fragmentation pattern for unknown product A. (B) Mass spectral fragmentation pattern for authentic product 9. (C) Mass spectral fragmentation pattern observed for unknown product B, which eluted at the retention time expected for epoxide 9, from the product mixture obtained in the CYPΔ2E1-catalyzed oxidation of 2-norcarene.

Figure 4

Portions of GC traces of products from oxidation of 2-norcarene (A) and 3-norcarene (B) with toluene monooxygenase from P. stutzeri OX1 (ToMO), soluble methane monooxygenase from M. capsulatus (Bath) (sMMO), and cytochrome P450 2B1 (CYP2B1). The identities of some of the products are indicated with the compound numbers.

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