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Chemical site-selective functionalization of proteins with boronic acids
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Chemical conjugation of a biomolecule, such as a peptide or a nucleic acid, with a functional molecule, for example a drug or a fluorophore, yields a bioconjugate, a polyfunctional entity that combines the properties of its components to elicit a biological effect. This kind of assemblies have found many successful applications, allowing the precise imaging of biological processes and delivering innovative treatments to the clinic, especially in oncology. The success of this class of constructs is determined by their ability to deliver the payload of choice to specific targets inside a biológical environment thanks to the targeting ability of the biomolecule. In order to have a functional conjugate, the payload must be linked to the targeting unit without altering the structure and function of the latter, preserving its ability to interact with the biological target. As a consequence, bioconjugation reactions must be performed in aqueous environment at physiological pH, avoiding strong reagents that might compromise the integrity of the biomolecule. Moreover, this ligation needs to be stable in physiological conditions while allowing the release of the payload in response to a stimulus that characterizes the biological target (pH, oxidative stress, enzymes).
Boronic acids are a class of biocompatible reagents with the ability to form reversible covalent ligations in water, leading to their successful application in the development of stimulus-responsive bioconjugates.
In this thesis, the 3-hydroxy quinolinone (3HQ) scaffold was studied as potential ligand for boronic acids in bioconjugation conditions, with the objective of using it as a functional unit for the reversible ligation of boronic acids to proteins. This scaffold was optimized for the desired purpose, yielding a structure that was successfully applied for the construction of a non-internalizing fluorescent bioconjugate. The resulting conjugate was tested in vitro, confirming its ability to deliver a borylated fluorescent probe to HT-29 cancer cells by targeting the 67 Laminin receptor.
Additionally, the 3HQ motif was explored as multivalent modulator of human phenylalanine hydroxylase (hPAH), a protein whose mutation is the cause of phenylketonuria (PKU). In this study, various assays were performed to assess the ability of 3 series of derivatives to stabilyze the structure of hPAH and to increase its catalytic activity. Finally, the best performing compounds from this study were docked onto the structure of hPAH to gain additional insight on their activity.
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Boronic acids Bioconjugation Reversible ligations Heterocyclic scaffolds Biological chemistry