Published: April 29, 2011 r2011 American Chemical Society 7853 dx.doi.org/10.1021/ja200551y | J. Am. Chem. Soc. 2011, 133, 7853–7858 ARTICLE pubs.acs.org/JACS Predictable Stereoselective and Chemoselective Hydroxylations and Epoxidations with P450 3A4 Aaron T. Larsen, Erin M. May, and Karine Auclair* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montr eal, Qu ebec, Canada H3A 2K6 b S Supporting Information ’ INTRODUCTION Natural systems are well recognized for streamlining the synthesis of large and complex molecules by iterative CÀC bond formation reactions followed by tailoring oxidations at selective positions. Although dramatic progress has recently been made, such a strategy is not yet generally available to chemists, the main difficulty being the poor selectivity achievable for oxidations at inactivated CÀH bonds. In particular, the stereoselective hydro- xylation of one methylene group in the presence of similar methylenes is probably the most challenging synthetic transfor- mation at the present time. Recent successes by chemists in this area include the regio- and stereoselective hydroxylation of pleuromutilin 1 and the total synthesis of deoxyerythronolide B by late-stage CÀH oxidations. 2 The successful methodology uses a bulky, electrophilic iron catalyst and hydrogen peroxide as the oxidant but typically suffers from overoxidation and limited functional group tolerance. The main difficulty arises in the fact that selective hydroxylation of tertiary carbons is favored over reactions at methylene groups. Recent advances in the hydro- xylation of inactivated CÀH bonds have thus focused on reactions at tertiary sp 3 carbons. 3À7 The electron-rich nature of tertiary CÀH bonds and their relative rarity in comparison to methylene and methyl carbons in natural products have made them excellent targets for metal-catalyzed hydroxylation. To our knowledge, however, no strategy using chemical catalysts has been successful at achieving predictable hydroxylation at one inactivated methylene CÀH bond among others of similar electronic properties. Enzymes remain unsurpassed at this task. Not only are enzymes typically regio-, chemo-, and stereoselec- tive, but they often perform well in mild aqueous conditions with innocuous oxidants, providing an environment-friendly alterna- tive to organometallic reagents. Monooxygenases such as cytochrome P450 enzymes (P450s or CYPs) arguably hold the greatest unexploited synthetic potential of any family of enzymes. 8 P450s are ubiquitous enzymes with major roles in mammalian drug metabolism, steroid biosynthesis, and bacterial biosynthesis of secondary metabolites, which are an exceptional source of pharmaceutical agents. Chemists have a special appreciation for P450s because of their impressive ability to catalyze selective hydroxylations at inactivated CÀH bonds. To date, however, this potential has remained highly untapped. Commercial applications using P450 enzymes are currently rare and limited to using whole cells. 9À13 Synthetic applications of P450s have been hindered mainly by the need for expensive cofactors, low turnover, and poor stability. These limitations have recently been partly overcome by rational and random mutagenesis, 14À16 yet the use of free enzymes remains nonideal for industrial processes. Research applications of P450s are more accessible considering the smaller scale. Unlike industrial settings, research environments require versa- tile and promiscuous, yet controllable, catalysts. Mutagenesis has mostly been used to modify the substrate specificity of P450s, 14À16 but rarely to increase their substrate promiscuity. 17 Although numerous P450s are highly promiscuous, product predictions are speculative at best. This problem is of special concern for drug-metabolizing P450s and has beleaguered the pharmaceutical industry for half a century. 18 The availability of a versatile catalyst for regio-, chemo-, and stereoselective hydroxylations of inactivated methylene groups would create a paradigm shift in synthetic chemistry. Such an advance would however require that the catalyst show reasonable Received: January 19, 2011 ABSTRACT: Enantioselective hydroxylation of one specific methylene in the presence of many similar groups is debatably the most challenging chemical transformation. Although chemists have recently made progress toward the hydroxylation of inactivated CÀH bonds, enzymes such as P450s (CYPs) remain unsurpassed in specificity and scope. The substrate promiscuity of many P450s is desirable for synthetic applications; however, the inability to predict the products of these enzymatic reactions is impeding advancement. We demon- strate here the utility of a chemical auxiliary to control the selectivity of CYP3A4 reactions. When linked to substrates, inexpensive, achiral theobromine directs the reaction to produce hydroxylation or epoxidation at the fourth carbon from the auxiliary with pro-R facial selectivity. This strategy provides a versatile yet controllable system for regio-, chemo-, and stereoselective oxidations at inactivated CÀH bonds and demonstrates the utility of chemical auxiliaries to mediate the activity of highly promiscuous enzymes.