OPPORTUNITIES AND CHALLENGES OF BIOCATALYSIS IN PHARMACEUTICAL SCIENCES: DEVELOPING TAILORED BIOCATALYSTS FOR SYNTHETIC APPLICATIONS

Biocatalysis has emerged as a powerful and sustainable alternative to traditional chemical synthesis, particularly in the preparation of stereochemically complex pharmaceutical intermediates. This doctoral research focuses on the development of tailored biocatalysts, specifically amine transaminases (ATAs), for the synthesis of pharmaceutically relevant compounds. To overcome typical limitations of wild-type enzymes, such as insufficient operational stability and narrow substrate scope, two complementary strategies were applied: enzyme immobilization and protein engineering. First, a biocatalytic cascade combining the ThDP-dependent enzyme Ao:DCPIP OR with stereoselective ATAs was established for the synthesis of (1S)-nor(pseudo)ephedrine analogues. The cascade was further optimized by covalent enzyme immobilization, enabling enhanced stability, efficient reuse, and integration into continuous-flow processes. This resulted in improved productivity, excellent stereoselectivity, and a more sustainable synthetic route. A second enzymatic workflow was developed for the synthesis of piperidine scaffolds, key intermediates in bioactive molecules. After identifying suitable ATAs and optimizing reaction conditions, the enzyme was immobilized and the process was transferred to a packed-bed flow reactor, achieving high conversion and diastereomeric excess. Finally, the (S)-selective Sbv333-ATA was biochemically and structurally characterized, revealing notable thermostability and solvent tolerance. Site-directed mutagenesis led to variants with an expanded substrate scope toward sterically demanding amines. Directed evolution efforts aimed at improving asymmetric amination are currently ongoing. Overall, this work highlights how biocatalysis is consolidating its role as an essential resource in modern synthetic chemistry. Technological advances are progressively shifting the paradigm from the passive use of naturally available enzymes to the proactive creation of optimized catalysts tailored for specific needs. In this perspective, the results achieved in this thesis provide a valuable contribution to the development of next-generation biocatalytic processes for the synthesis of chiral amines and exemplify the transformative potential of enzyme tailoring for the future of chemical manufacturing.