Crystallization-Assisted Asymmetric Synthesis of Enantiopure Amines Using Membrane-Immobilized Transaminase.

Abstract

The production of active pharmaceutical ingredients (APIs) requires enantiopure chiral amines, for which greener synthesis processes are needed. Transaminases (TAs) are enzymes that catalyze the enantioselective production of chiral amines from prochiral ketones through transamination under mild conditions. Yet, industrial applications of biocatalytic transamination remain currently hindered by the limited stability of soluble enzymes and by the unfavorable thermodynamic equilibrium of targeted asymmetric reactions. Enzyme immobilization can be applied to address stability, recoverability, and reusability issues. In the perspective of process intensification, we chose to immobilize TAs on polymeric (polypropylene) membranes. In the asymmetric synthesis of (R)-2-fluoro-α-methylbenzylamine ((R)-FMBA), such membrane-immobilized TAs exhibited superior specific activity and stability compared with soluble TAs; they also outperformed TAs immobilized on resins. The reaction yield remained, however, limited by thermodynamics. To further enhance the synthesis yield, the reaction was coupled with the in situ crystallization of (R)-FMBA with 3,3-diphenylpropionic acid (DPPA). By doing so, the theoretical equilibrium conversion was pushed from ∼44% to ∼83%. In fact, a 72% overall recovery yield of crystallized (R)-FMBA was demonstrated. The enantioselectivity of the reaction mixture was preserved. Importantly, purification was greatly facilitated since the target enantiopure amine was readily recovered as high-purity (R)-FMBA:DPPA crystals. The biocatalytic membranes were found to be fully reusable, performing successive high-yield asymmetric syntheses with only minor deactivation. Overall, the crystallization-assisted strategy proposed herein offers a greener path for the biocatalytic production of valuable chiral targets.