ArtiSt: An organocatalyst inspired by TPP installed in a steroid-carrying protein creates an artificial Stetterase.

The various methods (protein expression, mass spectrometry, chemical synthesis, protein design, engineering, product analysis) presented in this chapter were used to create the first artificial Stetterase constructed from an otherwise inactive protein scaffold and a synthetic thiamine pyrophosphate-inspired organocatalyst. The N-heterocyclic carbene (NHC)-derived organocatalyst was covalently incorporated into engineered protein scaffolds, creating an artificial "Stetterase" (ArtiSt). Cysteine residues were strategically introduced into selected scaffolds to enable site-specific bioconjugation of a synthetic NHC bearing a maleimide handle. Scaffold selection and mutagenesis were guided by structural analysis and computational modelling. Recombinant scaffold expression, purification, and optimization of functionalization conditions, generated hybrid catalysts that were evaluated using an intramolecular Stetter reaction as a model transformation. The NHC-functionalized ArtiSt catalyzed the reaction under ambient, aqueous conditions with low catalyst loading, affording modest yields and enantioselectivities. Activity was enhanced more than 20-fold by adopting a more stable protein scaffold, highlighting the critical role of the protein environment in controlling the catalytic behavior of the embedded organocatalyst. The modular platform described here establishes a foundation for optimizing activity and enantioselectivity and provides a general strategy for expanding NHC-mediated transformations within protein environments.