Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Biocatalysis in Drug Design: Engineered Reductive Aminases (RedAms) Are Used to Access Chiral Building Blocks with Multiple Stereocenters.

Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure-activity-property-toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery. A reductive aminase (RedAm) was engineered following a structure-guided mutagenesis strategy to produce the desired isomer. The engineered RedAm (IR-09 W204R) was able to generate the (S,S,S)-isomer 3 in 45% conversion and 95% ee from the racemic ketone 2. Subsequent palladium-catalyzed deallylation of 3 yielded the target primary amine 4 in a 73% yield. This engineered biocatalyst was used at preparative scale and represents a potential starting point for further engineering and process development.

Evaluation of chromatographic parameters in supercritical fluid chromatography of amino acids as model polar analytes and extended to polypeptide separations.

Chromatographic parameters have been evaluated regarding the supercritical fluid chromatography of amino acids as model compounds for polar and chargeable substances of biochemical and biopharmaceutical interest. Their inherent hydrophilicity requires suitable chromatographic conditions regarding stationary and mobile phase to obtain suitable solubility and peak performance. Ten stationary phases differing in polarity and type of ligands including neutral and charged ones were investigated regarding selectivity, peak performance and complementarity/orthogonality. The mobile phase composition regarding solvent and type of buffer additives by varying the degree of hydrophobicity of the acids and bases and degree of substitution in case of the basic amines. Addition of water to the methanol solvent was furthermore found crucial for obtaining a high peak performance. Some complementary selectivity could be obtained depending on a choice of column. Temperature and pressure variations basically only influenced retention with no selectivity changes. Suitable conditions obtained for the amino acids were then successfully applied to oligo- and polypeptides enabling a separation of 4 polypeptides and differing in only one of the 39 amino acids, thereby mimicking analysis of biotransformation reaction products. The utilization of water as mobile phase additive and sulfonic acids as buffer agents could serve as a generic methodology for polar and charged compounds such as amino containing biomolecules.