Krol, EdwardLee, Jeremy2020-11-032020-11-032020-102020-11-03October 20http://hdl.handle.net/10388/13123Alpha-synuclein aggregation is a hallmark pathological feature of Parkinson’s disease, and as such, it is thought to be a contributing factor to the disease. In the past, our group identified several compounds (1-aminoindan, nicotine, caffeine) capable of interacting with alpha-synuclein. These compounds were combined into bifunctional agents, such that caffeine was linked to one of 1-aminoindan, nicotine, or caffeine by a six-carbon chain. The nicotine and 1-aminoindan bifunctional agents were able to interact with alpha-synuclein in vitro and rescue cell growth in a yeast cell model overexpressing alpha-synuclein. However, the specifics of this protein-ligand interaction are unknown as alpha-synuclein is intrinsically disordered. To gain insight into possible binding regions on alpha-synuclein, we propose to use diazirine photoaffinity labelling to identify these sites. Our approach toward creating the diazirine probes was to link a four-carbon chain containing a diazirine to the amines of theophylline, nornicotine, and 1-aminoindan. We began by converting the ketone moiety of 4-hydroxy-2-butanone to a diazirine ring in three steps. The linker was then either tosylated or iodinated at the alcohol position. At this stage, the tosylated linker can be used to alkylate the N7-position of theophylline, and the iodinated linker can be used to alkylate the primary amine of 1-aminoindan, the secondary amine of nornicotine, or the bifunctional compounds to complete the synthesis of the diazirine probes. Isothermal titration calorimetry was used to determine all compounds binding affinity for alpha-synuclein; however, the methodology was unable to produce reliable binding data. Characterization of the diazirine probe’s activity toward alpha-synuclein aggregation was carried out using a Thioflavin T assay. The results from these studies were compared to the unlabelled compounds to determine the probe’s ability to inhibit alpha-synuclein aggregation relative to the original compounds. No direct anti-aggregation activity was observed for any diazirine-functionalized probes or unfunctionalized compounds. To investigate the binding interactions between alpha-synuclein and the photoaffinity probe, the probe was incubated with alpha-synuclein and irradiated with UVA for 30 minutes prior to trypsin digestion and analysis by electrospray mass spectrometry or tandem mass spectrometry. This final step resulted in labelling of alpha-synuclein by the caffeine-diazirine derivative at the glutamic acid 28 and tyrosine 39 amino acid positions. Labelling was not observed for the 1-aminoindan, nicotine, or caffeine six carbon linked caffeine diazirine compounds in the given conditions.application/pdfDiazirineAlpha-synucleinPhotoaffinity labellingThesis2020-11-03