Sinteza fluorescentnih termosenzorjev, pripravljenih s konjugacijo dveh fluoroforov

Pavlič, Maša (author), Pajk, Stane (mentor)

Luminiscenca je pojav, ki opisuje emisijo svetlobe, ki jo emitira snov potem, ko je predhodno absorbirala svetlobo nižje valovne dolžine, razdelimo pa jo na fluorescenco in fosforescenco. Predvsem fluorescenco izrabljajo številne tehnike, ki so postale nepogrešljive na področju bioznanosti. Bolj kot z opremo smo pri tehnikah, ki izrabljajo pojav fluorescence, omejeni z naborom ustreznih sond. Te največkrat pripravimo iz že preizkušenih fluoroforov, tako da nanje uvedemo ustrezne funkcionalne skupine. Prav omenjena »funkcionalizacija« predstavlja velik izziv, saj moramo optimizirati novo sintezno pot. V prvem delu magistrske naloge smo se osredotočili na pripravo derivatov fenola, iz katerega bi lahko nadalje sintetizirali kumarine, derivate nilsko rdečega ali nilsko modrega. Največ težav smo imeli z odščito aromatske metoksi skupine z BBr3, reakcija namreč ni potekla do želenih produktov ali pa so bili izkoristki zelo majhni. Namesto metilnega smo poskusili uvesti benzilni eter, vendar v tem primeru nekatere druge reakcije, npr. sinteza 1,2-dihidrokinolina, niso potekle po naših pričakovanjih. Nadalje smo poskusili uvesti funkcionalne skupine na kumarin 6 z uvedbo sulfonil kloridne skupine, ki bi omogočila pripenjanje aminov. Težavno stopnjo je predstavljalo klorsulfoniranje, za katerega predvidevamo, da je poteklo neselektivno na več mest kumarina 6, poleg tega so bili produkti zelo slabo topni. Na koncu smo se posvetili še uvedbi elektron privlačne ciano skupine na mesto 4 kumarina, za premik emisije v rdeči del spektra. Testirali smo, ali bi lahko reakcijo izvedli z manj toksičnim I2 namesto Br2, a se je slednji izkazal za boljšega. Nato smo poskušali z uvajanjem z estrom substituiranega azetidina oz. piperidina na 1-(benziloksi)-3-bromobenzen, a reakcija ni potekla. Bolj smo bili uspešni pri sintezi derivatov nilsko modrega. Tako smo pripravili spojino PMP-28, ki ima prosto amino skupino. Želeli smo pripraviti podobno spojino, a z boljšimi optičnimi lastnostmi, vendar smo v zadnji stopnji kot topili uporabili metanol in etanol, kar je prosti karboksilni skupini spojine PMP-34 pretvorilo v ustrezna estra. Slednjih nismo uspeli cepiti, ne da bi pri tem razpadel tudi fluorofor. V zadnjem delu magistrske naloge smo se osredotočili na sintezo fluorescenčnih sond za meritev temperature. Z optimizacijo pogojev smo sintetizirali dva kumarinska fluorofora PMP-37 in PMP-38, ki sta s propargilno skupino omogočila reakcijo »klik« z azidom rodamina B do PMP-39 in PMP-40. Z naraščanjem temperature namreč emisija rodamina B pada, emisija kumarina pa narašča.

• Language: Slovenian
• Keywords: fluorescenca, kumarin, rodamin, fluorescentna sonda
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-116855
• Publication date in RUL: 13.06.2020

Secondary language

• Language: English
• Title: Synthesis of fluorescent thermosensors prepared by conjugating two fluorophores

Abstract

Luminiscence describes the emission of light of a certain species after absorption of light at lower wavelengths and it can be divided in fluorescence and phosphorescence. Fluorescence is used in lots of indispensable techniques in bioscience. More than with equipment we are limited with range of suitable probes in this kind of techniques. Those probes are usually prepared from previously tested fluorophores by introducing suitable functional groups. This »functionalisation« presents a challenge as we must optimize new synthesis routes. In the first part of this master’s thesis we were focused on preparing phenol derivatives to further synthesize coumarins, nile red and nile blue derivatives. The biggest problem was deprotection of aromatic methoxy group with BBr3 as the reaction did not proceed to wanted products or the yields were low. Instead of methyl ester we also tried with benzyl ester but in this case other reactions, such as synthesis of 1,2-dichloroquinoline, did not occur as expected. We tried introducing functional groups on coumarin 6 with sulfonyl chloride group, which would enable linking different amino groups. The biggest challenge was chlorosulfonation which probably occurred non-selectively on various binding sites of coumarin 6. Products were also poorly soluble. In the end we would introduce an electron withdrawing -CN group on position 4 of coumarine to shift the emission spectra into red. We tested if the reaction could be performed with less toxic I2 instead of Br2, but Br2 showed as a better option. Later, we tried introducing ester-substituted azetidine or piperidine on 1-(benzyloxy)-3-bromobenzene, but the reactions did not occur. We were more successful with the synthesis of nile blue derivatives, thus we prepared PMP-28 which has a free amino group. We wanted to synthesize a similar compound but with better optical properties, however, in the last step we used methanol and ethanol as solvents, which converted the free carboxyl groups of the compound PMP-34 into the corresponding esters. We were not able to split the latter without the fluorophore falling apart. In the last part of this master's thesis, we focused on the synthesis of fluorescent probes for temperature measurement. By optimizing the conditions, two coumarin fluorophores PMP-37 and PMP-38 were synthesized, which enabled “click” reaction with their propagyl group and rhodamine B azide to create PMP-39 and PMP-40. As the temperature rises, rhodamine B emission drops and coumarin emission rises.

• Keywords: fluorescence, coumarin, rhodamine, fluorescent probe