Engineering of Reductive Aminases for Asymmetric Synthesis of Enantiopure Rasagiline

Abstract

Reductive aminases (RedAms) for the stereoselective amination of ketones represent an environmentally benign and economically viable alternative to transition metal-catalyzed asymmetric chemical synthesis. Here, we report two RedAms from Aspergillus calidoustus (AcRedAm) and bacteria (BaRedAm) with NADPH-dependent features. The enzymes can synthesize a set of secondary amines using a broad range of ketone and amine substrates with up to 97% conversion. To synthesize the pharmaceutical ingredient (R)-rasagiline, we engineered AcRedAm through rational design to obtain highly stereoselective mutants. The best mutant Q237A from AcRedAm could synthesize (R)-rasagiline with >99% enantiomeric excess with moderate conversion. The features of AcRedAm and BaRedAm highlight their potential for further study and expand the biocatalytic toolbox for industrial applications.

Keywords: chiral amine; rasagiline; rational design; reductive aminase; site saturation mutagenesis.

FIGURE 1

Phylogenetic tree of AspRedAm, AtRedAm, and potential RedAms. Four major clades were observed, in which four sequences from distinct clades were selected as candidates. The start sequences and candidate sequences are highlighted in green and red, respectively.

FIGURE 2

Selected ketones (A) and amines (B) for the substrate screening of RedAms. (C) RedAm-catalyzing reductive amination using selected substrates to access different amines. Reaction conditions: ketone/aldehyde (5 mM), amine (1–50 eq.), RedAm (1 mg ml⁻¹), NADP⁺ (1 mM), GDH (0.7 mg ml⁻¹), D-glucose (30 mM), and Tris–HCl buffer (100 mM, pH 9.0), 25°C, 220 rpm, 24 h. Conversion determined by HPLC or GC-FID analysis.

FIGURE 3

(A) Homology model of the AcRedAm dimeric structure in complex with NADPH (gray) and the iminium intermediate of 9e (green) incorporated by docking. (B) The binding pocket of AcRedAm is shown, which is at the dimer interface (red, polar; white, hydrophobic; magenta, exposed). (C) The residues located within 8 Å of the iminium intermediate are shown as sticks. Carbon atoms of subunits A and B are shown in cyan and orange, respectively.

FIGURE 4

Relative activity and ee values of saturation variants from site 207 (A), 214 (B) and 237 (C) of AcRedAm for the synthesis of rasagiline. Mutant W207D formed insoluble inclusion bodies. NA indicates no activity; ND indicates not determined for ee value. The activity of the wild-type enzyme was set as 100%. Error bars represent the standard deviations of three replicates.

FIGURE 5

Binding pockets (red, polar; white, hydrophobic; magenta, exposed) and distance between the reactive carbon atom of 9e and the hydride donating carbon (C4) of the nicotinamide of NADPH. (A) AcRedAm; (B) AcQ237A; (C) AcW207C; (D) AcW207S/Y214C. NADPH, (R)-rasagiline, and (S)-rasagiline are shown in gray, blue, and yellow, respectively.

FIGURE 6

Relative activity and ee values of the double-point mutants for rasagiline synthesis. The activity of wild-type enzyme was set as 100%. Error bars represent the standard deviations of three replicates.

FIGURE 7

Time course of the reactions. Conversion over time to rasagiline was measured with HPLC and GC (AcRedAm green, Q237A red, BaRedAm blue). Conditions: 5 mM 1-indanone 9, 250 mM propargylamine e, RedAm 1 mg/ml, 100 mM glucose, 1 mM NADP⁺, and 0.7 mg/ml glucose dehydrogenase. Reactions were incubated at 25°C, 220 rpm.