Enabling novel organocatalytic reactions in protein scaffolds through protein engineering

Hatton, Lewis 2025. Enabling novel organocatalytic reactions in protein scaffolds through protein engineering. PhD Thesis, Cardiff University. Item availability restricted.

	PDF - Accepted Post-Print Version Restricted to Repository staff only until 29 January 2027 due to copyright restrictions. Download (14MB)
	PDF (Cardiff University Electronic Publication Form) - Supplemental Material Restricted to Repository staff only Download (134kB)

Abstract

Cyclic secondary amines facilitate enamine and iminium ion catalysis, enabling the modification of carbonyl compounds. By integrating these catalysts into protein scaffolds, novel hybrid organocatalytic artificial enzymes with high activity and selectivity can be created. This thesis explores the selectivity and scope of such artificial enzymes, focusing on secondary amine-mediated iminium ion catalysis in protein environments. In chapter 2, the selectivity of the previously developed DHFR_Ala7DPK catalyst, which mediates hydride transfer using NADPH as a hydride source, was examined. Using chiral GC, we found a moderate preference for the S-enantiomer, which increased at higher pH. Under acidic conditions, however, the reaction produced a racemic mixture. Beyond hydride transfer, iminium ion catalysis offers a potential route for site-selective protein labelling. In chapter 3, our goal was to assess the reactivity of amino acid side chains with iminium ion catalysis. We achieved this by engineering the DHFR_Ala7DPK variant, initially identifying a position within the scaffold suitable for selective labelling of a reactive cysteine residue, followed by systematic mutation of this residue to every other amino acid. Our findings revealed promising labelling of tyrosine, tryptophan, arginine, and threonine residues. In chapter 4, we demonstrate the application of this system by introducing reactive secondary amines at various positions within an antibody affinity protein, leveraging its natural interaction with IgGs to facilitate iminium ion formation near nucleophilic residues on the antibody surface. By exploring multiple incorporation sites, we aimed to optimise reactivity and selectivity. Among the variants tested, the mutant SpA_N43_THP demonstrated the ability to modify the heavy chain at pH 8.0. This observed activity serves as a starting point for further refinement via directed evolution. Ultimately, this work lays the foundation for developing artificial enzymes capable of highly efficient, site-selective protein labelling.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Chemistry
Date of First Compliant Deposit:	29 January 2026
Last Modified:	29 Jan 2026 15:47
URI:	https://orca.cardiff.ac.uk/id/eprint/184277

Actions (repository staff only)

Edit Item