Sinteza okoljsko občutljivih fluorescenčnih sond na osnovi stirila, primernih za označevanje bioloških membran

Ramšak, Helena (avtor), Pajk, Stane (mentor)

Kemijske in funkcionalne spremembe celičnih membran so pogosto razlog za razvoj patogeneze različnih bolezni. Z razvojem fluorescence in STED (angl. Stimulated Emission Depletion) mikroskopije nam je omogočen vpogled in vivo v obnašanje in odzivanje celic na spremembe okolja. S tem namenom se povečuje potreba po sintezi fluorescenčnih sond, ki imajo ustrezne lastnosti, da se vgrajujejo v lipidni dvosloj membran. Prav tako je pri načrtovanju sond pomembno, da se njihove lastnosti odzivajo na spremembe v celičnih membranah. V okviru magistrske naloge smo sintetizirali fluorescenčne sonde, ki bi bile primerne za označevanje celičnih membran in opazovanje pod fluorescenčnim mikroskopom s tehniko STED. Načrtovali smo sintezo dveh tipov barvil, pri čemer smo se zaradi poteka sintez osredotočili na derivate s stirilno osnovo. Načrtovali smo sintezo barvil, ki so izhajala iz istega osnovnega skeleta, njihove lastnosti pa smo hoteli modificirali z uvajanjem različnih funkcionalnih skupin na osnovno strukturo. Sinteze so že v samih začetnih stopnjah povzročale težave, ki pa smo jih poskušali odpraviti s spreminjanem reakcijskih pogojev in reagentov. Za nekatere neuspešne reakcije nam je uspelo najti rešitve in optimizirati postopke, pri nekaterih pa vzroka nismo našli in sintezo opustili. Uspelo nam je sintetizirati dva končna produkta 11 in 13E na osnovi stirilne strukture. Praktično uporabnost barvil smo preizkusili na Institutu Jožefa Stefana. Opravili smo meritve tako s STED, kot tudi konfokalnim mikroskopom, prav tako pa smo opazovali tudi odziv emisijskega spektra spojin v različnih topilih. Zaradi svojih amfifilnih lastnosti sta se sondi uspešno vgradili v celično membrano, vendar je spojina 11 takoj povzročila apoptozo celic. Tudi pri meritvah s spojino 13E smo opazili znake apoptoze, vendar precej kasneje kot pri spojini 11. Na IJS smo ugotovili, da imata, z izjemo celične toksičnosti, sondi ustrezne lastnosti za snemanje spektrov s STED mikroskopom. Prav tako smo sintetizirali spojino, ki bi na kinolinski obroč imela pripet oksazol, vendar nadaljnje reakcije, s katerimi smo na oksazolni obroč želeli pripeti različne funkcionalne skupine, niso potekle, razloga za to pa nam ni uspelo odkriti.

• Jezik: Slovenski jezik
• Ključne besede: Fluorescenca, STED mikroskopija, membranske sonde, barvila na osnovi stirila
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-144352
• Datum objave v RUL: 16.02.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis of styryl-based environment-sensitive fluorescent probes, suitable for labelling of biological membranes

Izvleček

Chemical and functional changes in cell membranes can often be the cause of the development of various diseases. With the continuous development of fluorescence microscopy, especially STED microscopy, we are now able to gain insight into the relationship between the behavior of cells and the changes in their membrane and environment. To observe these changes with the fluorescence microscope, we need to synthesize fluorescent probes with suitable properties. It is important that these probes respond to changes in cellular properties and can be easily incorporated into cell membranes. In this master's thesis, we attempted to synthesize fluorescent probes suitable for observing cell membranes with the STED microscope. Originally, we planned to synthesize two types of dyes; however, our main focus was to prepare derivatives with a styryl base. We planned to modify the properties of the probes by introducing different functional groups into the styryl base structure. The synthesis presented problems at the initial stage, but we tried to overcome them by changing the reaction conditions and reagents. The synthesis of only two probes was successful: 11 and 13E. To test the practicality of these probes, we analyzed them at the Jožef Stefan Institute. We performed measurements using both STED and a confocal microscope and observed the response of the emission spectrum of these probes in different solvents. Due to their amphiphilic properties, both probes were incorporated into the cell membrane, however probe 11 immediately induced cell apoptosis. Signs of apoptosis were also detected in the measurements with compound 13E, but much later than with compound 11. At the IJS, we found that both probes, with the exception of cytotoxicity, had suitable properties for recording spectra with a STED microscope. We also synthesized a compound with an oxazole attached to the quinoline ring. With further reactions, we wanted to attach different functional groups to the oxazole, but the reactions did not proceed. We could not figure out the reason for the failed synthesis, so we were forced to forgo further progress in the originally planned reaction scheme.

• Ključne besede: Fluorescence, STED microscopy, membrane probes, styryl-based dyes