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

Abstract

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 enzymecatalyzed 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, structureguided 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%.

Emerging Technologies for Biocatalysis in the Pharmaceutical Industry

“…Another limitation of RedAm enzymes is their stability under process conditions, which is frequently reported to be problematic. − Mutations that stabilize the active IRED dimer are thought to play an important role in improving the performance of these enzymes, and stabilizing mutations can affect enzyme performance as much as mutations that affect substrate binding and turnover . This highlights a general problem for industrial biocatalysis: natural enzymes are usually not adapted to the high substrate concentrations and harsh conditions required in process chemistry, and this may limit their entrance into the biocatalytic reaction toolkit.…”

“…The speed with which RedAms have been incorporated into the process biocatalysis toolkit has been facilitated by close collaborations between academia and industry. Such collaborations accelerate the transfer of technology from academia to industry and are key to helping bridge the gap between enzyme classes known in nature and enzyme classes that are practical to implement at large scale. ,,,,− The success of the collaborative development of the IRED/RedAm suites of biocatalysts highlights the need for the pharmaceutical industry to support the search for new enzymes. Other biocatalytic platforms with high potential value for the pharmaceutical industry are those that target common functional groups in molecules and make new chiral centers.…”

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

“…Importantly, biocatalytic synthesis can play a crucial role in drug discovery since enzymes are tunable catalysts that allow for excellent control of chemoselectivity, regioselectivity and enantioselectivity. ,, Imine reductases (IREDs) are one emerging platform of biocatalyst − that perform a variety of reactions, including cyclic imine reduction, ,, reductive amination (RedAms), ,, and alkene reduction (EneIRED). , Interestingly, individual IREDs can behave simultaneously as an IRED, a RedAm, or an EneIRED depending on the substrate used while still maintaining excellent chemoselectivity …”

Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms

“…1a; Aleku et al, 2017;Gilio et al, 2022). These 'reductive aminases' (RedAms; Aleku et al, 2017;Gilio et al, 2022) have now been applied in the multikilogram (Schober et al, 2019) and ton (Kumar et al, 2021) scale synthesis of pharmaceutical precursors by many industrial (Schober et al, 2019;Kumar et al, 2021;Ma et al, 2021) and academic (Mangas-Sanchez et al, 2020;Yang et al, 2021;Zhang et al, 2022;Chen et al, 2023;Gilio et al, 2023) groups. RedAms were first identified in enzymes from fungi, and structures of two of them, AspRedAm from Aspergillus oryzae (Aleku et al, 2017) and AtRedAm from A. terreus (Sharma et al, 2018), each in complex with the NADP + cofactor and substrate or product molecules, have been published by our group.…”