Author: Kymberley Scroggie
Scroggie, Kymberley, 2020 Methods to incorporate silicon for the direct fluorination of proteins, Flinders University, College of Science and Engineering
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The direct fluorination of native proteins is a notoriously difficult task. The aqueous environments required by proteins severely hampers fluorination while the dry, organic environments that promote fluorination denature the proteins. To circumvent these issues, indirect fluorination methods whereby a prosthetic group is first fluorinated and then conjugated to a protein have become common place. However, when it comes to the radiofluorination of proteins these indirect methods are not well suited to the short half life of the fluorine 18 radionuclide (110 minutes). This doctoral thesis explores the incorporation of silicon, an inorganic fluoride acceptor, into proteins as a means towards their direct fluorination.
This thesis reports the design and synthesis of silanol derivatives and their incorporated into proteins via site selective chemical modifications at cysteine and lysine residues. The chemically modified proteins were found to undergo direct fluorination at the new incorporated silicon via substitution of the silanol hydroxide group. The direct fluorination proceeds in 100% aqueous conditions in time frames compatible with the half life of the fluorine 18 radionuclide. These results support the use silanols for the direct, aqueous fluorination of proteins and represents, for the first time, their use for this purpose.
This thesis also reports the site selective incorporation of a silanol into proteins via amber codon suppression. Using an existing tRNA and tRNA synthetase pair a silanol based unnatural amino acid was incorporated into green fluorescent protein. With a minimal incorporation efficiency, future work aimed at increasing the incorporation efficiency through the evolution of a tRNA and tRNA synthetase pair specific for the silanol based unnatural amino acid is required. Nevertheless, this research provides strong evidence for the continual investigation into the use of amber codon suppression for the incorporation of silanols into proteins.
The results presented within this thesis provides a constructive basis for further exploration into the use of silanols for the direct and aqueous fluorination of biomolecules. The results of this work give valuable insights into using silanols for the late stage radiofluorination of biomolecules and could lead to future advancements in the targeted and early detection of disease within the field of nuclear medicine.
Keywords: PET, fluorination, silicon, amino acid, amino acid incorporation, protein modification,
Subject: Chemistry thesis
Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Associate Professor Justin Chalker