Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.
The synthesis of steroid drugs by multistage modifications of the steroidal core is challenging since site-specific and selective modification is essentially required, which is often difficult or complicated for chemocatalysis. For example, the synthesis of Trenbolone (3), a versatile anabolic−androgenic steroid, relies on a four-step chemical procedure on its core modifications of estra-4,9-diene-3,17-dione (1). Here, we have designed a two-step chemoenzymatic strategy that includes a biocatalytic one-pot C11-hydroxylation/17β-ketoreduction of 1 with a computationally designed P450 monooxygenase and an appropriate 17-ketosteroid reductase to generate 11α-OH-9(10)dehydronandrolone (2a) as an intermediate followed by chemical dehydration to introduce the double bond at carbons 11 and 12 with the formation of Trenbolone (3). To obtain a highly active and C11-selective enzyme, molecular dynamics simulations were performed, uncovering a crucial role of water molecules for substrate recognition and targeted hydroxylation of steroids. Moreover, Trenbolone is further subjected to esterification to produce Trenbolone acetate (9) that has been widely used in veterinary medicine. Finally, our approach enables the regio-and stereoselective synthesis of both steroid drugs 3 and 9 on a (nearly) gram scale with 83−91% isolated yields, showing great potential for industrial applications.
Steroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio-and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.
Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.
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