Understanding the Speciation of Ruthenium Arene Complexes as Precursors to Unnatural Enzyme Cofactors

Ruthenium arene complexes have been extensively explored as metallo-pharmaceuticals and as small molecule catalysts. Exploring the overlap between these areas, this thesis describes a body of work aimed at quantitatively understanding the biological speciation and catalytic behaviour of ruthenium arene complexes when exposed to the many potential Lewis basic ligands provided by protein scaffolds. Combining nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography and other biophysical characterisation techniques, the speciation of ruthenium arene bipyridine complexes with small molecule amino acids, short peptides and whole proteins has been monitored. A 19F NMR spectroscopic method was developed to quantitively trace the preferred amino acid binding partners of ruthenium complexes coordinated to fluorinated ligands. Liquid chromatography mass spectrometry (LC-MS) was used to explore the ligand exchange behaviour between ruthenium complexes and protein scaffolds, particularly variants of the small proteins ubiquitin and cytochrome b562. Additionally tandem MS/MS experiments were used to determine the final protein binding sites of non-fluorinated ruthenium fragments coordinated to proteins. The resulting deep understanding of how ruthenium arene complexes coordinate to specific proteins was used to develop artificial metalloenzymes (ArMs) with direct protein-metal coordination. The catalytic capabilities of these hybrid systems was then explored. ArMs are synthetic biocatalysts that result from either the combination of an artificial metallo-cofactor being introduced into a protein scaffold or a natural metalloprotein being evolved to perform catalytic reactivity. Taking inspiration from naturally occurring metalloproteins, this research showed that it was possible to form ArMs via a ligand exchange process between a ruthenium arene precursor complex and a protein scaffold, resulting in the precursor complex being activated towards catalysis. Direct protein – metal coordination enables the protein to impart both an electronic and steric contribution to catalysis and attenuate reactivity at the metal centre in ways that have not been previously studied. The four helical bundle protein cytochrome b562 was selected as the protein scaffold for ArM development due to its dynamic structure and nascent haem binding site, which in the absence of haem provides a hydrophobic pocket capable of accommodating a ruthenium cofactor and catalytic substrate. Cytochrome b562 – ruthenium hybrids (with direct coordination) were identified that have catalytic transfer hydrogenation activity greater than a known dimeric catalyst. This demonstrates an exciting starting point to explore the evolutionary potential of these ArMs through directed evolution, hopefully enhancing catalytic activity.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Biggs, George

Advisors dc:contributor.advisor

• Barker, Paul
• Boss, Sally

• Artificial Metalloenzymes
• Ruthenium
• Ligand Exchange

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e30c34de-fe24-4be0-ac0c-d5bb85ea4d17/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-6526-9236
0000-0003-0094-0054
0000-0002-6194-1889

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/318020