20.500.12556/RUL-182695 Optimizacija sintezne poti in priprava derivatov fluorescenčnih bisbenzimidazolnih označevalcev DNA Optimization of the synthetic pathway and preparation of derivatives of fluorescent bisbenzimidazole DNA labels V magistrski nalogi smo se osredotočili na optimizacijo sintezne poti do bisbenzimidazolnega označevalca DNA Hoechst 33258 ter na pripravo njegovih derivatov za nadaljnjo funkcionalizacijo s fluorofori. Hoechst 33258 se specifično veže na z adeninskimi in timinskimi baznimi pari bogata zaporedja v malem žlebu DNA, vendar je njegova uporaba pri mikroskopiji živih celic omejena zaradi vzbujanja v ultravijoličnem območju. Kljub temu so Hoechstova barvila zaradi dobre prepustnosti skozi plazemsko membrano in učinkovitega označevanja DNA v živih celicah nepogrešljiva pri fluorescenčni mikroskopiji. Njihova emisijska intenziteta se ob vezavi na DNA izrazito poveča, kar je posledica rigidizacije bisbenzimidazolnega skeleta in spremembe polarnosti okolja; to v praksi pomeni manjši signal ozadja in boljše razmerje med signalom in šumom. Tako kot večina spojin, ki interagirajo z DNA, so tudi Hoechstova barvila toksična, vendar je njihova toksičnost pri uporabljanih koncentracijah sprejemljiva in bistveno manjša kot pri sondah, ki se interkalirajo med bazne pare. Njihova glavna pomanjkljivost ostaja ekscitacija s fototoksično UV-svetlobo, zato bi bili analogi, ki bi jih bilo mogoče vzbujati z vidno svetlobo in bi imeli večji Stokesov premik, za raziskovalno delo zelo koristni. Glavni namen naloge je bil optimizirati sintezno pot do Hoechstovega označevalca, pripraviti njegove strukturne analoge z možnostjo nadaljnje funkcionalizacije ter razviti nove sonde z izboljšanimi fotofizikalnimi lastnostmi. V eksperimentalnem delu smo ponovili večstopenjsko sintezo bisbenzimidazolnega ogrodja Hoechst 33258 in optimizirali posamezne reakcijske korake z namenom izboljšanja izkoristkov. Najzahtevnejši korak je bil tvorba druge benzimidazolne enote, pri čemer se je po več poskusih kot najprimernejša izkazala kondenzacija med diaminom in aldehidom. Z opisano reakcijo smo uspešno uvedli različne funkcionalne skupine in pripravili serijo novih sond. Optične lastnosti spojin smo ovrednotili z merjenjem ekscitacijskih in emisijskih spektrov v prisotnosti in odsotnosti DNA. Ob vezavi na tarčo se je fluorescenčni signal povečal, pri nekaterih derivatih pa smo opazili tudi premik spektra v vidno območje. Biološko vrednotenje na celični liniji metastatskega pankreasnega adenokarcinoma je pokazalo, da nekatere nove sonde učinkovito prehajajo celično membrano in selektivno označujejo celično jedro. In this master’s thesis, we focused on the optimization of the synthetic route to the bisbenzimidazole DNA label Hoechst 33258 and on the preparation of its derivatives for further functionalization with fluorophores. Hoechst 33258 binds specifically to the minor groove of DNA at AT-rich sequences, but its application in live-cell imaging is limited by excitation in the ultraviolet region. Nevertheless, Hoechst dyes remain indispensable in fluorescence microscopy because they readily cross the plasma membrane and efficiently stain DNA in living cells. Their fluorescence intensity increases markedly upon binding to DNA, which is a consequence of rigidization of the bisbenzimidazole scaffold and changes in the polarity of the surrounding environment; in practice, this results in lower background signal and an improved signal-to-noise ratio. Like most compounds that interact with DNA, Hoechst dyes are to some extent toxic, but at the concentrations used their toxicity is acceptable and significantly lower than that of probes that intercalate between base pairs. Their main drawback remains excitation with phototoxic UV light; therefore, analogues that could be excited with visible light and exhibit a larger Stokes shift would be highly valuable for research applications. The main aim of this thesis was to optimize the synthetic route to the Hoechst label, prepare its structural analogues suitable for further functionalization, and develop new probes with improved photophysical properties. In the experimental part, we repeated the multistep synthesis of the bisbenzimidazole scaffold of Hoechst 33258 and optimized individual reaction steps in order to improve yields. The most demanding step was the formation of the second benzimidazole unit, where condensation between a diamine and an aldehyde proved to be the most suitable approach after several attempts. Using this reaction, we successfully introduced various functional groups and prepared a series of new probes. The optical properties of the compounds were evaluated by measuring excitation and emission spectra in the presence and absence of deoxyribonucleic acid. Upon binding to the target, the fluorescence signal increased, and in some derivatives a spectral shift toward the visible region was also observed. Biological evaluation in a metastatic pancreatic adenocarcinoma cell line showed that some of the newly prepared probes efficiently crossed the cell membrane and selectively stained the cell nucleus. bisbenzimidazol fluorescenca Stokesov premik Hoechst DNA sonda bisbenzimidazole fluorescence Stokes shift Hoechst DNA probe true false false [U. Golob] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-05-21 08:45:08 2026-05-21 08:45:24 2026-05-22 11:04:52 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.1:535.37(043.2) 125345 279049987 32189.pdf 32189.pdf 1 2ED1248B302F206653D80098F4D69927 aab796680cb5a15957a805ced887e6156c1065b3d0e79e97c3f88bf583c0198d 5723b7dc-54e0-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=234174 Fakulteta za farmacijo 0 0 0