20.500.12556/RUL-170204 Rational incorporation of emergent fluorinated groups into amino acids and target peptides Načrtna vgradnja uveljavljajočih se fluoriranih funkcionalnih skupin v aminokisline in ciljne peptide The introduction of fluorine or fluorinated functional groups has become a key strategy in agrochemical and pharmaceutical development. In peptide chemistry, fluorination has been shown to enhance metabolic stability, stabilize desired secondary structure and provide insight into biological interactions via $^{19}$F NMR spectroscopy. Despite their promising properties, chalcogen-associated fluorinated groups remain underexplored in the peptide arena. Among these, the trifluoromethylthio (CF$_3$S) group has emerged as a privileged substituent due to its lipophilicity profile and strong electron-withdrawing properties. In this study, we developed an efficient method for direct C(sp$^2$)–H trifluoromethylthiolation of aromatic amino acids (tryptophan, tyrosine, DOPA), as well as their biologically relevant monoamine analogs, such as serotonin, dopamine and tyramine. The developed method enabled the late-stage incorporation of the CF$_3$S moiety into tryptophan-containing peptides and facilitated the gram-scale synthesis of enantiopure Fmoc-protected building blocks (77–93 % yield). The latter were utilized in SPPS of model peptides and endomorphin-1 analogs. Furthermore, the local hydrophobic effect of the CF$_3$S group when incorporated into the peptide was investigated. A substantial increase in hydrophobic interactions was revealed, showing the potential of site-selective trifluoromethylthiolation for improving the drug-like properties of peptides. As part of this work, a library of fluorinated endomorphin-1 (EM1) analogs was synthesized, featuring various CF$_3$(S)-based modifications. A total of 10 ligands were evaluated in vitro for μ- and δ-opioid receptor binding affinity and functional activity, exhibiting comparable binding affinities (up to 1.38 nM K$_i$) to the parent EM1. In the final part of the dissertation, the selective oxidation of CF$_3$S-amino acids to obtain corresponding trifluoromethyl sulfoxide and sulfone derivatives was investigated. The oxidized building blocks were successfully incorporated into peptides via SPPS, and their physico-chemical properties were analyzed. Additionally, we have developed a high-yielding synthetic route to protected perfluoro-tert-butyl serine analogs via Mitsunobu reaction. The results presented in this doctoral dissertation advance the methodology for the incorporation of the CF$_3$S group and its higher oxidation congeners into amino acids and target peptides via SPPS or late-stage functionalization. The conducted physicochemical and biological activity studies enhance our understanding of the impact of sulfur-associated fluorinated motifs on peptide properties and pave the way for further application of emergent fluorinated functional groups in the design of peptide-based therapeutics or biomaterials. Uvedba fluora ali fluor-vsebujočih funkcionalnih skupin je danes ena od ključnih strategij pri razvoju agrokemikalij in farmacevtikov. Načrten vnos fluorovih atomov v peptidne sisteme omogoča izboljšano metabolično stabilnost, stabilizacijo želenih sekundarnih struktur in vpogled v biološke interakcije s pomočjo $^{19}$F NMR spektroskopije. Navkljub izredno obetavnim lastnostim ostajajo fluorirani motivi, asociirani s halkogeni relativno neraziskani na področju peptidne kemije. Med tovrstnimi motivi se je trifluorometiltiolna (CF$_3$S) skupina uveljavila kot privilegiran motiv zaradi svoje lipofilnosti in močnega elektron privlačnega učinka. V sklopu te študije smo razvili učinkovito metodo za neposredno C(sp$^2$)–H trifluorometiltioliranje aromatskih aminokislin (triptofan, tirozin, DOPA) ter njihovih biološko pomembnih monoaminskih analogov, kot so serotonin, dopamin in tiramin. Razvita metoda omogoča vnos CF$_3$S motiva v stranske verige triptofan-vsebujočih peptidov v pozni stopnji ter pripravo enantiomerno čistih Fmoc-zaščitenih gradnikov v gramskem merilu (77–93 % izkoristek). Le-ti so bili uspešno uporabljeni v sintezi modelnih peptidov in analogov endomorfina-1 (EM1) na trdnem nosilcu (SPPS). V nadaljevanju smo raziskali doprinos CF$_3$S skupine k lokalni hidrofobnosti stranskih verig peptidov. Izkazano znatno povečanje hidrofobnih interakcij kaže na potencial načrtnih CF3S-modifikacij za izboljšavo farmakokinetičnih parametrov peptidov. V sklopu raziskav je bila sintetizirana knjižnica fluoriranih analogov EM1 z različnimi CF$_3$(S) modifikacijami farmakoforjev. Serija ligandov je bila in vitro ovrednotena glede na afiniteto vezave na μ- and δ-opioidna receptorja ter njihovo funkcionalno aktivnost. Najbolj obetavni ligandi so izkazali primerljivo afiniteto vezave (do 1,38 nM K$_i$) starševskemu peptidu EM1. V zadnjem sklopu disertacije smo preučevali selektivno oksidacijo CF$_3$S-vsebujočih aminokislin za sintezo ustreznih trifluorometil sulfoksidov in sulfonov. Oksidirane gradnike smo uspešno uporabili v sintezi peptidov na trdnem nosilcu ter analizirali vpliv substituentov na fizikalnokemijske lastnosti peptidov. Poleg tega smo razvili učinkovito sintezno pot do zaščitenega perfluoro-terc-butil serina z uporabo ključne Mitsunobu reakcije z visokimi izkoristki. Pridobljeni rezultati v sklopu doktorskega dela predstavljajo napredek v razvoju metodologije za vgradnjo CF$_3$S skupine ter višje oksidiranih motivov v aminokisline in tarčne peptide s SPPS ali funkcionalizacijo v pozni stopnji. Izvedene študije fizikalno-kemijskih lastnosti in biološke aktivnosti izboljšujejo naše razumevanje vpliva fluoriranih motivov, asociiranih z atomom žvepla, ter izpostavljajo visok potencial uveljavljajočih se fluoriranih funkcionalnih skupin pri načrtovanju novih zdravilnih učinkovin ali biomaterialov na osnovi peptidov. amino acids peptide chemistry trifluoromethylthiolation late-stage functionalization endomorphin-1 SPPS hydrophobicity oxidation of sulfides $^{19}$F NMR spectroscopy aminokisline peptidna kemija trifluorometiltioliranje funkcionalizacija v pozni stopnji endomorfin-1 SPPS hidrofobnost oksidacija sulfidov $^{19}$F NMR spektroskopija true false false Angleški jezik Slovenski jezik Doktorsko delo/naloga 2025-07-02 15:15:35 2025-07-02 15:15:35 2025-07-25 09:38:36 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 23024 243814403 Gregorc_Jure_-_Nacrtna_vgradnja_uveljavljajocih_se_fluoriranih_funkcionalnih_skupin_v_aminokisli.pdf Gregorc_Jure_-_Nacrtna_vgradnja_uveljavljajocih_se_fluoriranih_funkcionalnih_skupin_v_aminokisli.pdf 1 DFEEA442C53E3F39A2A357F69F1CD0FD 142fdc29b8d4ba10d2d170b2df140dda63571d0a8b62d0805887dc020f59f968 c00125ad-5744-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=212157 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0