Rational incorporation of emergent fluorinated groups into amino acids and target peptides

Gregorc, Jure (author), Iskra, Jernej (mentor), Brigaud, Thierry (comentor)

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.

• Language: English
• Keywords: amino acids, peptide chemistry, trifluoromethylthiolation, late-stage functionalization, endomorphin-1, SPPS, hydrophobicity, oxidation of sulfides, $^{19}$F NMR spectroscopy
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-170204
• COBISS.SI-ID: 243814403
• Publication date in RUL: 02.07.2025

Secondary language

• Language: Slovenian
• Title: Načrtna vgradnja uveljavljajočih se fluoriranih funkcionalnih skupin v aminokisline in ciljne peptide

Abstract

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.

• Keywords: aminokisline, peptidna kemija, trifluorometiltioliranje, funkcionalizacija v pozni stopnji, endomorfin-1, SPPS, hidrofobnost, oksidacija sulfidov, $^{19}$F NMR spektroskopija