well-studied of all known cytochromes P450. While wild-type CYP101 can oxidize dichloro- and trichlorobenzenes to chlorophenols, it is not able to metabolize the more highly substituted pentachlorobenzene (7) and hexachlorobenzene (8). Polychlorinated phenols do not present a significant hazard, as they are degraded by microorganisms in the environment. Thus, finding or constructing an enzyme that would metabolize highly chlorinated aromatics such as 7 and 8 would represent a significant advance toward a solution. Once constructed, the genes encoding the CYP101 system could be genetically incorporated into chlorophenol-degrading microorganisms to convert chlorobenzenes to chlorophenols that would then be further degraded by the host.39 The authors' challenge then was to bioengineer CYP101 so it would be able to hydroxylate 7 and 8 to form 9 (Scheme 1). Reasoning that changes that increased hydrophobicity and decreased the size of the active site would be beneficial to that end, three specific mutations were introduced. The mutant enzyme was not only able to metabolize 7 and 8, but exceeded the hydroxylase activity of wild type by three orders of magnitude.
If cytochrome P450-catalyzed hydroxylation of benzylic or allylic C-H bonds proceeds by the same mechanism operative for the simple saturated systems discussed above, they would be expected to be favored processes. Resonance stabilization of a radical intermediate would lower the activation energy for both benzylic and allylic hydroxylation. This is generally what is found in drug metabolism studies.
White et al.40 reinvestigated the stereochemistry of hydroxylation of the prochiral benzylic carbon atom of phenylethane (10), using enantiomerically pure (R)- and (S)-phenylethane-1-d as substrates (Scheme 2). General findings of particular note were (1) hydroxylation occurs almost exclusively, greater than 99%, at the benzylic position to give the isomeric a-methylbenzyl alcohols (11), a result consistent with the benzylic position being a favored site of attack, and (2) the percentage yield of the minor metabolites, 2-phenylethanol (12) and 4-ethylphenol, (13), more than triples when phenylethane-1-d2 is used as the substrate. The isotopically driven switching to other sites of metabolism not only indicates the operation of a significant isotope effect but the tripling of such metabolites, particularly 4-ethylphenol, also indicates that the substrate has considerable freedom of motion within the active site and the potential to form multiple catalytically productive active site binding orientations.8'41'42 It is not uncommon for a single cytochrome P450 to catalyze the formation of multiple regioisomeric products from the same substrate.43,44 where the primary metabolite is often formed by oxidation at the energetically most favored position.45
An early example of allylic hydroxylation was provided by Licht and Coscia,46 who reported that the CYP2B 4-catalyzed hydroxylation of the terpenes geraniol (14) and nerol (16) occurred almost exclusively at the C-10 (E)-methyl group of both compounds (eqns  and ). What is informative about this example is that the adjacency of a double bond converts the methyl group to a major site of oxidative attack.
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