Expanding the biocatalytic toolbox: Novel reactivities and engineering of dehydrogenases Applications in the synthesis of carboxylic acids derivatives and the chemo-enzymatic construction of APIs

This thesis explores the versatile application of oxidoreductases, specifically alcohol dehydrogenases (ADHs), to expand the biocatalytic toolbox through novel methodologies and chemoenzymatic cascades. The research is divided into two primary trajectories: uncovering non-natural enzyme reactivity and integrating known biocatalysts into multi-step synthesis.
The first part investigates ADHs for unconventional transformations. Beyond the traditional reduction of ketones—highlighted by the characterization of two rare, anti-Prelog selective NAD-dependent ADHs, this work establishes ADHs as potent oxidative catalysts. By manipulating reaction environments and employing site-directed mutagenesis, new synthetic pathways were developed for the production of amides, thioesters, and esters from alcohols, amines, and thiols. Furthermore, a sustainable, single-enzyme methodology for the oxidation of primary alcohols to carboxylic acids was developed, utilizing acetone as a sacrificial substrate for cofactor regeneration. The oxidative potential of ADHs was further demonstrated in a continuous flow system using immobilized enzymes on glass beads to achieve selective esterification.
The second part focuses on complex molecule synthesis via integrated catalysis. This includes an enantioselective chemoenzymatic cascade combining transaminases with organocatalysis, and the design of chimeric enzymes fused with formate dehydrogenase for streamlined cofactor regeneration in flow reactors. Finally, a nine-step retrosynthetic analysis of the antihypertensive API dilevalol is presented, combining eight enzymes with chemocatalytic steps.
Collectively, these findings demonstrate that the catalytic plasticity of ADHs, enhanced by protein engineering and flow chemistry, provides green and efficient alternatives to traditional synthetic organic chemistry.