Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Abstract

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active site residues of the cytochrome P450 from Bacillus megaterium (P450-BM3), we have engineered a highly active His-ligated variant of P450-BM3, BM3-Hstar, that can be employed for the enantioselective synthesis of the levomilnacipran core. This enzyme catalyzesthe cyclopropanation of N,N-diethyl-2-phenylacrylamide (1) with an estimated initial rate of over 1000 turnovers per minute and can be used under an aerobic environment. Cyclopropanation activity is highly dependent on the electronics of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity.

Gram‐Scale Synthesis of Chiral Cyclopropane‐Containing Drugs and Drug Precursors with Engineered Myoglobin Catalysts Featuring Complementary Stereoselectivity

“…[7] Reflecting this notion, cyclopropanation biocatalysts that can reliably offer complementary stereoselectivity have so far remained unavailable. [3, 5, 8] Here, we report the development and characterization of a panel of engineered Mb catalysts that give access to trans -( 1R,2R ) configured 1-carboxy-2-aryl-cyclopropanes with high selectivity and catalytic activity across a broad range of olefin substrates. We further demonstrate that these Mb-catalyzed reactions can be carried out and scaled up using whole cell systems.…”

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

“…Whole-cell biocatalysis employing CYP BM3 mutants has already been successfully applied for the biosynthesis of fine chemicals [56,[60][61][62][63][64], steroids [65], and drugs [3,66]. In the current study another example is presented by showing that CYP BM3 mutants can be used for efficient regio-and stereoselective biosynthesis of drug metabolites of NET, a steroid that has been widely used in combined hormonal contraceptives.…”

Inside Cover: Directed Evolution: Bringing New Chemistry to Life (Angew. Chem. Int. Ed. 16/2018)

“…Our lab took advantage of this inherent ability of a bacterial cytochrome P450 to evolve a highly efficient enzyme for production of the chiral ciscyclopropane precursor to the antidepressant medication levomilnacipran. [6] Our group and that of Rudi Fasan have since pushed a variety of heme proteins to synthesize other chiral cyclopropane pharmaceutical precursors, including one used in the synthesis of ticagrelor, a medication used to prevent the reoccurrence of heart attacks. [7] In our case, we identified a truncated globin from Bacillus subtilis, which catalyzes the reaction at low levels and also showed some selectivity for producing the single, desired diastereomer of the ticagrelor cyclopropane precursor from ethyl diazoacetate and 3,4-difluorostyrene (Figure 1).…”