title: By the powers combined: the design and study of artificial metalloenzymes as photocatalysts
Creators: name: Agrawal, Arush
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Photocatalysis
subject: Artificial metalloenzymes (ArM)
subject: Enantioselectivity
subject: Iridium(III) complexes
subject: Peptide-Ru ArM

description: Photocatalysis is a powerful technique allowing the synthesis of products not easily accessible via traditional thermal chemistry. Transition metal complexes represent a major class of photocatalysts, with cyclometallated complexes of iridium(III) and ruthenium(II) being particularly prominent. However, the field of photocatalysis suffers from the difficulties experienced in controlling the enantioselectivity of the final product. In nature, enzymes catalyze reactions yielding products with extreme enantioselectivities. However, their scope is limited to reactions found in nature and can only utilize metals which are bio-available and natural co-factors (or their analogues) of the enzymes. This hampers the range of chemistry that can be achieved. Artificial metalloenzymes (ArM) aim to bridge the gap between the two catalysis regimes, by combining novel metals with proteins, to enable new-to-nature reactions with the advantage of increased enantioselectivity. In the present work, two distinct methods were used to create novel photocatalytic ArMs and investigate their photophysical characteristics; introduction of an unnatural amino acid that capable of chelating Ir(III) into a naturally occurring protein and using coiled-coil peptide containing bipyridine sidechains which was designed de-novo to allow the binding of Ru(II). The first strategy relied on the introduction of the unnatural amino acid bipyridine alanine (BpyAla) containing a 2,2’-bipyridine (bpy) side chain to the human Sterol Carrier Protein (SCP-2L). The bipyridine side chain can act as a binding site for the Ir(III) ion. Initially, 4 residues (V83, A100, Q111 and M112) on the protein scaffold were mutated to BpyAla and [Ir(ppy)2(bpy)]+ (where ppy = 2-phenylpyridine) bound on those sites. This library of 4 ArMs was studied to determine their photophysical properties, particularly the absorbance, fluorescence, quantum yields and excited state lifetimes, and limited structural study was carried out via circular dichroism spectroscopy. All the Ir-ArM variants showed a blue shifted fluorescence spectra compared to free [Ir(ppy)₂(bpy)]⁺. Further, all 4 ArMs showed an excited state lifetime more than twice as long as the free complex, and as high as 632 ns in the case of A100Bpy-Ir(ppy)₂ variant (vs 43 ns for the free complex). The incorporation of the Ir(III) center into the protein also resulted in up to 15x higher quantum yields vs free complex in aqueous solutions. Having formed and studied the photophysical properties of the Ir-ArM, a photocatalytic reaction was attempted (the homodimerization of chalcone) using the designed metalloenzyme as the catalyst. While the observed enantioselectivity was not very high with any of the modified proteins, the N-gly-M112Bpy-Ir(ppy)2 variant gave the highest e.e. (10%) of those tested. While attempts were made to introduce different Ir(III) complexes with various substituted phenylpyridine ligands to the protein, they proved unsuccessful. Similar to enzymes found in nature, peptides can also be used to confer stereoselectivity in photocatalytic reactions. The second method of synthesising ArMs discussed in the following pages starts with de-novo designed peptides where the desired structural features can be incorporated at the design stage. Collaborators at the University of Bristol designed a hexameric coiled-coil peptide of the form A₃B₃ containing a hydrophobic tunnel, in which the peptide strand B contains BpyAla. Hydrophobic interactions pre-arrange the bipyridine residues in a manner which allows them to chelate Ru(II), to form an octahedral [Ru(bpy)₃]²⁺ complex, which is a well-known photocatalyst. The formation of the peptide-Ru ArM was followed by photophysical studies. When only peptide B was used to form [Ru(bpy)₃]²⁺ the excited state did not change appreciably form that observed for the free [Ru(bpy)₃]²⁺ complex in an aqueous solution. However, the coiled-coil system (PeptideA)₃(PeptideB)₃Ru(bpy)₃ showed a 7% longer lifetime vs [Ru(bpy)₃]²⁺. This system also showed drastically reduced quantum yield (0.6%) vs the free [Ru(bpy)₃]²⁺ complex (3.9%).