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.

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.

Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.

Biocatalytic Synthesis of Moclobemide Using the Amide Bond Synthetase McbA Coupled with an ATP Recycling System.

The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equiv of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2-phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine, and hexylamine, and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9H-ß-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its amidation conformation, coupled with modeling and mutational studies, reveal an amine access tunnel and a possible role for residue D201 in amine activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piperidine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobemide, through the reaction of 4-chlorobenzoic acid with 1.5 equiv of 4-(2-aminoethyl)morpholine, and utilizing polyphosphate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equiv of ATP, required for recycling of the cofactor.