Design and Exploration of Novel Organocatalytic Artificial Enzymes for Iminium Biocatalysis

The development of organocatalytic artificial enzymes has gained increasing interest in recent years, as researchers aspire to combine the broad reaction chemistry of organocatalysts with the efficiency and specificity of enzymes. The work described in this thesis aimed to develop novel organocatalytic artificial enzymes, specifically targeting iminium-type transformations. Since the selection of a biomolecular scaffold plays a central role in the design of artificial enzymes, different scaffolds were explored and functionalized with either a natural (lysine) or an unnatural catalytic residue (pAF and D/LProK). By comparing how these distinct amino acids perform in various biomolecular scaffolds, the thesis seeks to demonstrate how the choice of the catalytic residue and scaffold can significantly influence the activity and selectivity of the artificial enzyme. Overall, this thesis aims to shed light on different design principles of organocatalytic artificial enzymes and contribute to the creation of effective biocatalysts for asymmetric transformations. Therefore, Chapter 2 comprises a literature review on the most common biomolecular scaffolds employed in the design of artificial enzymes, focusing on their versatility and catalytic applications. Selected examples are described and categorized according to three different strategies used for generating artificial enzymes, followed by a comparison of these methods and our perspective. Chapter 3 discusses the exploration of novel biomolecular scaffolds for iminium biocatalysis. Specifically, scaffolds with open hydrophobic pockets and enzymes with described binding sites for the targeted substrate were investigated. Selected candidates were modified with amine groups such as lysine and ncAAs pAF or DProK and the potential of the novel artificial enzymes was evaluated in an asymmetric Michael addition reaction. Chapter 4 presents further research on the most promising candidates identified in Chapter 3, including the outcomes of a directed evolution campaign. A comparative analysis of selected variants was performed to investigate how specific mutations affect the catalytic activity in the model reaction. Chapter 5 focuses on an additional biomolecular scaffold in which its catalytic lysine was replaced by the ncAAs pAF and D/LProK. The catalytic efficiency of the resulting variants was evaluated in an asymmetric Michael addition reaction. Crystal structures of the artificial enzyme harboring pAF, free and in complex with the substrate, were obtained to investigate differences in active site architecture. Additionally, a mutagenesis study was conducted to expand the binding pocket of the biomolecular scaffold for a better accommodation of D/LProK. Finally, Chapter 6 summarizes the key findings and conclusions of this work, followed by future perspectives on artificial enzyme development for biocatalysis.