20.500.12556/RUL-159172 Priprava izbranih derivatov enaminona za detekcijo proteinskih agregatov Preparation of selected enaminone derivatives for the detection of protein aggregates Nastanek proteinskih agregatov znotraj celic lahko vodi do mnogih patoloških stanj, kot so Alzheimerjeva, Parkinsonova in Huntingtonova bolezen, amiotrofična lateralna skleroza (ALS), demenca Lewijevih telesc in frontotemporalna demenca. Ker se proteinski agregati pri zgoraj naštetih boleznih pojavljajo tudi leta pred simptomi, ponujajo možnost zgodnjega odkrivanja bolezni in njenega zdravljenja. Za detekcijo teh agregatov se lahko uporabljajo tudi fluorescenčne molekulske sonde na osnovi "push-pull" elektronskih sistemov. S tem namenom smo sintetizirali tri fluorescenčne molekulske sonde z enaminonskim fragmentom. Enaminon smo sintetizirali preko reakcije enolne oblike acetilne skupine z N,N-dimetilformamid dietil acetalom oziroma N,N-dimetilformamid dimetil acetalom. Vse tri sinteze so bile uspešno izvedene in spojine so bile okarakterizirane z 1H in 13C NMR ter IR spektroskopijo, masno spektrometrijo visoke ločljivosti (HRMS) in meritvijo temperature tališča. Čistost spojin smo preverili s tekočinsko kromatografijo visoke ločljivosti (HPLC). Spojinam smo tudi pomerili optične lastnosti, kot sta absorpcija, emisija in ekscitacija. Sledila je izvedba testov detekcije proteinskih agregatov. Kot eksperimentalni model smo uporabili protein TDP-43, katerega agregati so značilni za bolnike z ALS. Naš protein TDP-43 je imel na C-koncu fuzijskega partnerja vezavni protein za maltozo (MBP), ki je povečal topnost proteina in nam omogočal induciranje agregacije z dodatkom proteaze virusa jedkanja tobaka (TEV). Pokazali smo, da vse tri fluorescenčne sonde uspešno razlikujejo med agregirano in neagregirano obliko TDP-43. The formation of protein aggregates within cells can lead to many pathologic states ranging from Alzheimer's, Parkinson's, and Huntington's disease, amyotrophic lateral sclerosis, Lewy-body dementia to frontotemporal dementia. As these protein aggregates typically appear years before the onset of symptoms, they offer a path to an early diagnosis and treatment of disease. Detection of these aggregates can be achieved with fluorescent molecular probes, which function on the principle of push-pull electron systems. With this in mind, three fluorescent molecular probes with the enaminone moiety were synthesized. The enaminones were prepared with the reaction of the enol tautomer of the acetyl group with N,N-dimethylformamide diethyl acetal or N,N-dimethylformamide dimethyl acetal. All three probes were successfully synthesized and characterised with 1H, 13C NMR and IR spectroscopy, high resolution mass spectrometry (HRMS) and melting point measurements. The purity of the probes was proved by high-pressure liquid chromatography (HPLC). Also, the optical properties of the probes such as absorption, emission, and excitation were examined. Lastly, these probes were tested for their ability of aggregate detection. As our experimental model, the TDP-43 protein, associated with the amyotrophic lateral sclerosis (ALS) patients, was used. Our TDP-43 had a C-terminal fusion partner maltose binding protein (MBP), which allowed induction of aggregation with the addition of tobacco etch virus protease (TEV). It was shown that all three synthesised probes are able to differentiate between the soluble and aggregated form of TDP-43. proteinski agregati amiotrofična lateralna skleroza fluorescenčne molekulske sonde enaminon protein aggregate amyotrophic lateral sclerosis fluorescent molecular probe enaminone true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2024-07-02 12:16:58 2024-07-02 12:17:02 2024-07-13 03:30:29 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22873 201617923 Priversek_Maj_-_Priprava_izbranih_derivatov_enaminona_za_detekcijo_proteinskih_agregatov.pdf Priversek_Maj_-_Priprava_izbranih_derivatov_enaminona_za_detekcijo_proteinskih_agregatov.pdf 1 E472E81E32C0E8A94B63AA93881A6081 fbaeabd57108588f5a8f7061216b042b2423b50f476c58e26244ee88e768b965 388273b9-385c-11ef-926e-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=187105 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0