Campopiano, Dominic

Wallace, Stephen

Clarke, David

MacAulay, Alice

EastBio

2024-05-10T10:22:46Z

2024-05-10T10:22:46Z

2024-05-10

The application of biocatalysis in industrial synthesis continues to rise at pace, driven by a demand for sustainable synthetic methods. As a result, chemo-enzymatic cascades which merge the use of chemocatalysis and biocatalysis are of growing interest. Whilst the majority of enzymatic reactions take place in water, chemocatalysis is typically performed in organic solvents making solvent-compatibility an issue. One option to overcome this is the use of protein hosted organocatalytic reactions. Proteins are inherently compatible with aqueous solvents, but their internal environment contains hydrophobic pockets and functional groups suited to organocatalysis. Progress in protein engineering has enabled us to design, evolve and select structures which can incorporate and exploit the reactivity of non-proteinogenic components. Whilst modification of existing cofactors has increased organocatalytic reactivity and reaction scope, in some cases modification is not compatible with the native protein host. In this thesis we look to use an alternative protein scaffold covalently functionalised with an organocatalyst. We also consider how the chiral environment of the protein could be utilised to enable an enantioselective reaction. In this work we select, express and purify several protein scaffolds with cysteine (Cys) residues at selected positions to allow for functionalisation with an organocatalyst. For some of the scaffolds (e.g. human steroid carrier protein, hSCP) Cys-containing variants already exist. Whilst for others (e.g. Thermus thermophilus SCP, TTSCP), the placement of Cys residues was guided by structural analysis, docking (AutoDock Vina) and modelling of the constructs using AlphaFold. We functionalised our chosen scaffold with N-heterocyclic carbenes (NHCs), a large group of organocatalysts which have been inspired by the natural cofactor thiamine pyrophosphate (TPP). NHCs catalyses a wide number of reactions including C-C bond formation and other reactions that are not known in nature. We synthesised novel NHCs with appropriate handles to enable bioconjugation to specific positions in a protein scaffold. We went on to screen and select conditions for functionalisation of the protein scaffolds and identify the best-functionalised scaffolds to test as catalysts. We also investigated the use of genetic code expansion (GCE) for the incorporation of an NHC unnatural amino acids (UAAs) into a protein. To do this we synthesised novel NHC based UAAs and screen existing orthogonal translation systems. To test the catalytic activity of our functionalised protein scaffolds (hSCP and TTSCP) we used an intramolecular Stetter reaction as a well-studied model reaction. The Stetter reaction uses a nucleophilic catalyst to catalyse C-C bond formation between an aldehyde and an α,β-unsaturated carbonyl forming a 1,4 dicarbonyl product. This reaction is of interest as the synthesis of enantiopure 1,4 dicarbonyl compounds remains a challenging transformation in synthetic chemistry. Stetter reactions are mostly undertaken in organic solvents with only one, aqueous Stetter catalyst reported to date, which is not enantioselective. The TPPdependant Stetterase enzymes such as PigD and MenD can produce chiral products in water, however, these too have limitations in their substrate scope and soluble recombinant expression. By using a protein hosted organocatalyst we have the opportunity to incorporate NHCs with different core structures and enable more diverse chemistries. Here we show SCP scaffolds covalently functionalised with an NHC catalyse an intramolecular Stetter reaction with modest yields and % e.e. We demonstrate that our functionalised proteins operate under ambient conditions with low catalyst loading. Furthermore, we demonstrate that activity can be increased >20 fold by altering the protein scaffold. This is the first example of an artificial Stetterase constructed from an inactive protein scaffold and a synthetic organocatalyst. This breakthrough discovery lays the foundations to optimise the catalytic activity and improve the enantioselectivity. Furthermore, this proof-of-concept study paves the way for this methodology to be applied to other NHC/protein scaffold combinations.

https://hdl.handle.net/1842/41766

http://dx.doi.org/10.7488/era/4489

en

The University of Edinburgh

Stetterase

artificial Stetterase

organocatalyst

protein scaffold

biocatalysis

industrial synthesis

sustainable synthetic methods

chemo-enzymatic cascades

chemocatalysis

chiral environment

enantioselective reaction

cysteine (Cys) residues

human steroid carrier protein (hSCP)

Thermus thermophilus SCP, TTSCP

N-heterocyclic carbenes (NHCs)

thiamine pyrophosphate (TPP)

genetic code expansion (GCE)

unnatural amino acids (UAAs)

low catalyst loading

inactive protein scaffold

synthetic organocatalyst

Creation of an artificial Stetterase through the design, synthesis and installation of an organocatalyst into a protein scaffold

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy