Title BODIPY spojevi fluorescentni markeri za biomakromolekule i fototerapeutici Title (english) BODIPY Compouds Fluorescent Markers for Biomacromolecules and Phototherapeutics Author Katarina Zlatić Mentor Nikola Basarić (mentor)Mentor Nela Malatesti (komentor)Committee member Ivana Ratkaj (predsjednik povjerenstva)Granter University of Rijeka Defense date and country 2020-09-15, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Jedna od metoda liječenja raka je fotodinamička terapija koja se temelji na korištenju molekula koje apsorbiraju zračenje te prijenosom energije generiraju citotoksični singletni kisik. Međutim, stanice raka često su hipoksične što predstavlja glavnu
prepreku u uspješnosti terapije. U sklopu ovog doktorskog rada istraživan je drugačiji pristup u liječenju raka u kojem učinkovitost terapije ne ovisi o prisutnosti kisika. U sklopu ove doktorske disertacije sintetizirane su četiri klase novih
fluorescentnih BODIPY boja koje kao supstituent sadrže fotoreaktivnu skupinu iz koje se fotokemijskom reakcijom generiraju kinon-metidi (QM). BODIPY spojevi izvrsni su fluorofori zbog termičke i fotokemijske stabilnosti, apsorpcije vidljivog svjetla, velikih molarnih apsorpcijskih koeficijenata i kvantnih prinosa fluorescencije. Nadalje, QM-i su reaktivni intermedijeri u kemiji fenola koji reagiraju s nukleofilima, uključujući DNA i proteine. Stoga, fotopobuđivanjem netoksičnih BODIPY spojeva nastaju toksični QM-i. Fotofizička svojstva BODIPY spojeva istraživana su korištenjem stacionarne i vremenski ovisne fluorescencijske spektroskopije, dok je fotokemijska reaktivnost ispitivana osvjetljavanjem otopina spojeva u metanolu. Nastajanje produkata metanolize upućuje na nastajanje QM-a koji su detektirani korištenjem laserske pulsne fotolize. Primijećeno je i da fotogenerirani QM reagiraju s goveđim serumskim albuminom pri čemu se spojevi kovalentno vežu na protein, što se može primijeniti za vizualizaciju proteina u gel elektroforezi ili fluorescencijskoj mikroskopiji. Jedno od naših glavnih otkrića je da se fotoreakcije triju klasa spojeva odvijaju samo pri pobuđivanju u viša singletna pobuđena stanja, dok se iz S1 stanja deaktiviraju fluorescencijom. Ova neuobičajena anti-Kasha fotokemijska reaktivnost u potpunosti je razjašnjena računskim studijima na TD-DFT razini teorije. Koncept korištenja jedne valne duljine svjetlosti za fotoaktivaciju boje i vezanje spoja na protein te druge valne duljine za očitavanje fluorescencije vrlo je primjenljiv u razvoju fluorescentnih markera. Antiproliferativno djelovanje BODIPY boja istraženo je korištenjem MTT testa na humanim stanicama raka. Nakon tretiranja stanica BODIPY spojevima i zračenjem, neke su klase spojeva pokazale pojačanu aktivnost što ih čini potencijalnim lead molekulama za razvitak novih fototerapeutika u terapiji karcinoma.
Abstract (english) One method to treat cancer is photodynamic therapy, which is based on the use of molecules that absorb light and transfer excitation energy, forming reactive oxygen species responsible for the cytotoxic effects. However, cancer cells are often hypoxic causing obstacles in the success of the therapy. This PhD thesis features a new approach of cancer phototreatment that does not rely on oxygen. Four classes of new fluorescent BODIPY dyes were synthesized that
were substituted by photochemically reactive substituents, which in the photodeamination reaction deliver quinone methides (QMs). BODIPY dyes are excellent fluorophores owing to their thermal and photochemical stability, absorption of visible light, high molar absorption coefficients and fluorescence quantum yields. Furthermore, QMs are ubiquitous reactive intermediates in the chemistry of phenols that react with nucleophiles including DNA or proteins. Thus, photoexcitation of the BODIPY dyes, which are not cytotoxic, transforms them to cytotoxic QMs. Photophysical properties of the BODIPY dyes were investigated by steady-state and time-resolved fluorescence, whereas photochemical reactivity was probed by irradiations in methanol. Methanolysis products indicated formation of QMs, which were detected by laser flash photolysis.
Furthermore, we have shown that photogenerated QMs react with bovine serum albumin and induce covalent attachment to the protein, which can be used for the visualization in gel electrophoresis or microscopy. One of our major discoveries is that the photoreaction of four classes of molecules takes place only upon excitation to higher singlet excited states, whereas S1 states deactivate by fluorescence. This unusual anti-Kasha photochemical reactivity was fully elucidated by computations at the TD-DFT level of theory. This concept to use one wavelength of light to photoactivate the dye and attach it to protein, and other wavelength to read the fluorescence is highly applicable in the development of fluorescent sensors. Antiproliferative activity of the dyes was investigated by MTT tests on human cancer cells. Upon treatment of cells with the dyes and irradiation, some classes of dyes showed enhanced activity, rendering potential leads in the development of cancer phototerapeutics.
Keywords
BODIPY
kinon-metidi
fluorescentne boje
fotofarmaceutici
fototerapeutici
fotokemija
fluorescentno obilježavanje proteina
Keywords (english)
BODIPY
quinone methides
fluorescent dyes
photopharmaceuticals
phototerapeutics
photochemistry
fluorescent protein labeling
Language croatian URN:NBN urn:nbn:hr:193:068955 Promotion 2021 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Closed access Terms of use Created on 2021-12-16 13:02:45
