Small-molecule-based crosslinking and bioconjugation of biomolecules

Chapter 1: The chemical modification and bioconjugation of proteins is an expanding yet challenging area of chemical biology. The superior nucleophilicity of cysteine presents it as a uniquely reactive bioconjugation handle. Inefficient photoinitiated methods and enzymatic methods (formylglycine-generating enzyme) are known for the oxidation of the thiol moiety of cysteine into the corresponding aldehyde (formylglycine). In this chapter, a photoinitiated transformation of the thiol group of a cysteine residue into a bioconjugation handle using a small molecule is described. The efficient generation of this reactive bioconjugation handle could facilitate rapid and selective modification of cysteine residues within protein. Chapter 2: Benzo[1,2,4]triazin-7-ones have previously been shown to exhibit submicromolar cytotoxicity against breast and prostate cancer cell lines and strong correlations to the naturally occurring anti-cancer agent, pleurotin. Given that the incorporation of a pyridine moiety into cytotoxic compounds has also been shown to significantly alter cytotoxicity against cancer cell lines, two newly synthesized pyrid-2-yl benzo[1,2,4]triazin-7-ones have been evaluated for cytotoxicity by using the MTT assay and National Cancer Institute (NCI) COMPARE analysis. The stable free radical precursors of the pyrid-2-yl benzo[1,2,4]triazin-7-ones were also subject to cytotoxicity evaluation with comparisons made to TEMPO, a stable free radical known to exhibit cytotoxicity. Both stable free radical precursors showed specificity towards the prostate cancer cell line but were several orders of magnitude less cytotoxic than the corresponding pyrid-2-yl benzo[1,2,4]triazin-7-ones. Both pyrid-2-yl benzo[1,2,4]triazin-7-ones exhibited increased cytotoxicity against most cancer cell lines compared with the parent phenyl benzo[1,2,4]triazin-7-one analogue as shown by the NCI. NCI COMPARE analysis of the pyrid-2-yl benzo[1,2,4]triazin-7-ones showed strong correlations to the naturally occurring anti-cancer compound pleurotin. The discovery of anti-cancer activity and correlations to pleurotin for pyrid-2-yl benzo[1,2,4]triazin-7-ones presents these novel iminoquinones as potential anti-cancer agents. Chapter 3: The synthesis of alicyclic ring-fused [1,2-a]benzimidazoles has attracted attention as precursors to benzimidazolequinones, a known class of bioreductive anti-tumour agents. Current synthetic protocols often require the use of transition metal catalysts, high molar mass hypervalent iodine reagents and strong bases. The most convenient method involves the oxidative cyclization of o-cyclic amine substituted anilines using hydrogen peroxide and trifluoroacetic acid. In this chapter hydrogen peroxide and methanesulfonic acid were used to synthesize an azocane-fused[1,2-a]benzimidazole, which was further oxidized into the benzimidazolequinone. The incorporation of halogen atoms onto the quinone moiety has previously been shown to enhance cytotoxicity and provides the potential for these moieties as synthetically useful intermediates. In this work, the combination of hydrogen peroxide with hydrohalic acids (H2O2/HX) has been used to synthesize a new series of halogenated azocane-fused[1,2-a]benzimidazoles and benzimidazolequinones through a one-pot oxidative cyclization, halogenation and demethylation of o-cyclic amine substituted anilines. The cytotoxicity of a series of ring-fused[1,2-a]benzimidazolequinones has been evaluated using the MTT assay. The novel H2O2/HX protocol allows efficient generation of potent, anti-cancer ring-fused benzimidazolequinones. Chapter 4: Covalently crosslinked proteins, in long-lived connective tissues, are among the major modifications caused by the Maillard process. Advanced glycation end products (AGEs) accumulate over time and are generated to a greater extent in diabetes. Glucosepane is a major protein (lysine–arginine) crosslink, yet its mechanism of formation is poorly understood. Previous studies have suggested a key α-dicarbonyl intermediate, which is formed through a series of tautomerizations from the well-known Amadori product (derived from glucose and lysine). Cyclization of this intermediate with a proximal arginine residue yields glucosepane. In this work, isotopic labelling studies have been used to explore the possibility of an alternative mechanism for the formation of glucosepane, an intramolecular 1,5-hydride shift. The detection of a mass consistent with that of unlabelled glucosepane suggested that a 1,5-hydride shift does not occur from either the C6 position of the Amadori product to the C2 position or from the C5 position of the protonated imine intermediate to the C1 position. Elucidation of the formation pathways of these major protein crosslinks is a significant step for the development of effective therapeutics against the accumulation of AGEs in tissues.

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College of Science, National University of Ireland, Galway

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NUI Galway

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

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University of Galway Theses (PhD Theses)