Optimizacija sinteznih pogojev za pripravo fluoroforov s heterocikličnim skeletom z mostnim dušikovim atomom in vrednotenje odvisnosti njihove emisije od pH

Gorenc, Marija (author), Pajk, Stane (mentor), Kokot, Hana (comentor)

Fluorescentne spojine imajo v sodobni znanosti ključno vlogo pri označevanju celičnih struktur, sledenju biološkim procesom in razvoju diagnostičnih orodij. V okviru magistrske naloge smo se osredotočili na fluorofore s heterocikličnim skeletom z mostnim dušikovim atomom, predvsem so nas zanimali kot potenciali označevalci lizosomov. V okviru magistrske naloge smo najprej raziskali vpliv različnih kislin na izkoristek reakcije modelnega fluorofora s tiazolnim skeletom z mostnim dušikovim atomom (SP-66Z). Za spremljanje in kvantifikacijo produktov smo uporabili tekočinsko kromatografijo (HPLC), v izbranih primerih pa tudi tekočinsko kromatografijo sklopljeno z masno spektrometrijo (LC-MS). S slednjo smo uspeli najti intermediat, ki osvetljuje reakcijski mehanizem nastanka opisanih spojin. Nadalje smo s pomočjo UV-Vis in fluorescenčne spektroskopije preučili odvisnost optičnih lastnosti (absorbance in fluorescence) spojin od pH. Na podlagi teh rezultatov smo izbrali sondi SP-66Z in SP-80Z kot najprimernejši kandidatki za nadaljnje testiranja. Zadnji sklop naloge je bil usmerjen v testiranje izbranih fluoroforov na mišjih pljučnih epitelijskih celicah (LA-4) z uporabo različnih mikroskopskih tehnik, vključno z mikroskopijo življenjske dobe fluorescence oz. FLIM (ang. Fluorescence Lifetime Imaging Microscopy) in z mikroskopijo vzbujenega praznjenja emisije oz. STED (ang. Stimulated Emission Depletion) mikroskopijo. Cilj teh eksperimentov je bil oceniti uporabnost sintetiziranih spojin za označevanje lizosomov in na splošno oceniti njihove foto-fizikalne lastnosti. Kar se tiče označevanja lizosomov, obe sondi označita vezikle v celicah, vendar sondi nista kolokalizirali s komercialno sondo za lizosome SiR-lysosome. Kar se tiče foto-fizikalnih lastnosti, pa se je predvsem sonda SP-66Z izkazala za relativno fotostabilno, medtem ko je sonda SP-80Z hitro fotobledela. Poleg tega je sonda SP-80Z vplivala na obliko jeder, kar nakazuje na citotoksičnost sonde. Sonda SP-66Z se je izkazala tudi za občutljivo na okolico, saj se je življenjski čas fluorescence razlikoval glede na velikost veziklov kamor se je sonda vgradila. Lahko zaključimo, da glede na rezultate predvsem sonda SP-66Z predstavlja dobro izhodišče za nadaljnji razvoj sond za označevanje celičnih organelov.

• Language: Slovenian
• Keywords: fluorofori, pH odvisna fluorescenca, lizosomi, spektroskopija in mikroskopija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-169750
• Publication date in RUL: 07.06.2025

Secondary language

• Language: English
• Title: Optimization of synthesis conditions for preparation of fluorophores with a heterocyclic skeleton-containing a bridgehead nitrogen atom and evaluation of their emission dependence on pH

Abstract

Fluorescent compounds play a crucial role in modern science, particularly in labeling cellular structures, tracking biological processes, and developing diagnostic tools. In this master’s thesis, we focused on fluorophores with a heterocyclic scaffold containing a bridged nitrogen atom, primarily exploring their potential as lysosomal markers. Initially, we investigated the effect of various acids on the yield of a model reaction involving a thiazole-based fluorophore with a bridged nitrogen atom (SP-66Z). Liquid chromatography (HPLC) was used to monitor and quantify the reaction products, and in selected cases, liquid chromatography coupled with mass spectrometry (LC-MS) was employed. Using LC-MS, we identified an intermediate that provided insights into the reaction mechanism behind the formation of the studied compounds. Subsequently, we used UV-Vis and fluorescence spectroscopy to examine how the optical properties (absorbance and fluorescence) of the compounds depend on pH. Based on these results, SP-66Z and SP-80Z were selected as the most promising candidates for further testing. The final part of the thesis was dedicated to testing the selected fluorophores on murine pulmonary epithelial cells (LA-4) using various microscopy techniques, including Fluorescence Lifetime Imaging Microscopy (FLIM) and Stimulated Emission Depletion (STED) microscopy. The aim of these experiments was to assess the applicability of the synthesized compounds for lysosome labeling and to evaluate their overall photophysical properties. Regarding lysosomal labeling, both probes marked vesicles within the cells; however, neither showed colocalization with SiR-lysosome, a commercial probe for lysosomes. In terms of photophysical characteristics, SP-66Z demonstrated relatively high photostability, while SP-80Z exhibited rapid photobleaching. Additionally, SP-80Z appeared to affect nuclear morphology, indicating its cytotoxicity. SP-66Z also showed sensitivity to its environment, as its fluorescence lifetime varied depending on the size of the vesicles in which it was localized. In conclusion, based on the results obtained, particularly those related to SP-66Z, this compound represents a promising starting point for further development of probes for labeling cellular organelles.

• Keywords: fluorophores, pH-dependent fluorescence, lysosomes, spectroscopy and microscopy