Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio-and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio-and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming tradeoffs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450 BM3 mutants which enable nearly perfect regio-and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity−selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids.
A new spin: The addition of chemically inert perfluoro carboxylic acids (green; see picture) to P450 enzymes results in dramatic activation of their catalytic activity as a result of the conversion of the Fe/heme from a low‐spin to a high‐spin state, and the reduction of the binding‐pocket size. Together these effects allow otherwise inert substrates such as propane and even methane to be oxidized.
More than 7000 ee determinations are possible per day in the analysis of (+)/(−)‐cyclohexylethylamine 1. This result demonstrates the potential of capillary array electrophoresis (CAE) with chirally modified electrolytes for the determination of the enantiomeric purity of organic compounds.
A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.
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