Sinteza fluoroforov tipa donor-akceptor za detekcijo bioloških označevalcev : raziskovalni podatki, obravnavani v doktorskem delu

Rogan, Matic (author), Košmrlj, Janez (mentor), Urankar, Damijana (researcher), Pevec, Andrej (researcher), Knez, Damijan (researcher), Bresjanac, Mara (researcher), Blinc, Lana (researcher)

Alzheimerjeva bolezen (AB) je kronična nevrodegenerativna bolezen, pri kateri igra ključno vlogo agregacija amiloida β (Aβ) in proteina tau. Kljub napredku na področju imunokemijskih in slikovnih metod ostaja zgodnja, široko dostopna in cenovno ugodna diagnostika AB še vedno velik izziv. Ena izmed obetavnih možnosti so majhne fluorescenčne molekulske sonde, ki bi omogočale zaznavo bioloških označevalcev AB v telesnih tekočinah na osnovi merjenja fluorescenčnega odziva. V doktorskem delu smo se zato osredotočili na načrtovanje, sintezo in vrednotenje malih organskih fluoroforov tipa D–π–A za detekcijo agregatov, povezanih z AB. V okviru doktorskega študija smo pripravili štiri generacije paličastih fluoroforov, ki se razlikujejo po elektron donorski (D) in akceptorski (A) skupini ter dolžini oz. zgradbi π-konjugiranega sistema (π). Prva generacija obsega ketone, β-diketone, β-enaminone in malononitrile z benzenskim, naftalenskim, naftil-fenilnim in stilbenskim π-skeletom. Druga generacija temelji na različno substituiranih 2H-piran-2-onih, tretja generacija na 3-acetilnih in BF$_2$-β-diketonatnih 2H-piran-2-onih, četrta generacija pa na konjugiranih alkenih z nitro skupino. Fluorofore smo sintetizirali z uporabo različnih klasičnih in sodobnih organskih sinteznih metod, med drugim Suzuki–Miyaura in Heckova reakcija, Horner–Wadsworth–Emmonsova reakcija, Buchererjeve reakcija, Buchwald–Hartwigovega aminiranje, mešana Claisenova kondenzacija, Knoevenagelova kondenzacija, ciklizacija 2H-piran-2-onskega obroča ter Henryjeva reakcija. S tem smo pripravili strukturno raznolike generacije spojin, pri katerih smo lahko sistematično proučevali vpliv D, π in A na optične in vezavne lastnosti spojin. Optične lastnosti pripravljenih fluoroforov smo proučevali v topilih z različno polarnostjo, in sicer v heksanu, diklorometanu, acetonitrilu, metanolu in HEPES pufru. Pokazali smo, da na lege absorpcijskih in emisijskih maksimumov, Stokesove premike ter kvantne izkoristke pomembno vplivajo narava akceptorja A ter dolžina in zgradba π-konjugiranega sistema. Podaljšanje π-sistema in uvedba močnejših akceptorjev sta praviloma vodila do batokromnega premika absorpcije in emisije, v nekaterih primerih tudi v bližnje infrardeče območje. Pomemben del doktorskega dela je bilo tudi in vitro vrednotenje optičnih in vezavnih lastnosti pripravljenih fluoroforov v prisotnosti fibril Aβ$_{1-42}$. Rezultati so pokazali, da te lastnosti niso odvisne zgolj od dolžine π-konjugiranega sistema, temveč tudi od narave aromatskega skeleta in akceptorske ter donorske skupine. Pri več generacijah spojin smo ugotovili, da je mogoče z ustrezno kombinacijo teh strukturnih elementov doseči ugodno ravnotežje med afiniteto sond do fibril Aβ$_{1-42}$ in lego ter intenziteto emisijskih maksimumov po vezavi.

• Language: Slovenian
• Keywords: Alzheimerjeva bolezen, amiloid β, D–π–A, fluorofor, 2H-piran-2-on, nitroalken, optične lastnosti, vezavne lastnosti
• Typology: 2.20 - Complete scientific database of research data
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-185995
• Data col. methods: Self-administered writings and/or diaries; Observation:Laboratory; Experiment: Laboratory; Synthesis; Measurements and tests
• Publication date in RUL: 25.08.2026

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: English
• Title: Synthesis of donor-acceptor type fluorophores for the detection of Alzheimer's disease biomarkers : research data underlying the doctoral dissertation

Abstract

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder in which the aggregation of amyloid β (Aβ) and tau protein plays a key role. Despite advances in immunochemical and imaging methods, early, accessible, and cost-effective AD diagnostics remain challenging. One promising option is small fluorescent molecular probes that could detect AD biomarkers in body fluids by fluorescence measurements. This thesis focused on designing, synthesizing, and evaluating small organic D–π–A fluorophores for the detection of AD-associated aggregates. We prepared four generations of rod-like fluorophores, differing in donor (D), acceptor (A), and the length and structure of the π-conjugated system (π). The first generation comprises ketones, β-diketones, β-enaminones, and malononitriles with benzene, naphthalene, naphthyl–phenyl, and stilbene π-skeletons. The second generation is based on variously substituted 2H-pyran-2-ones, the third on 3-acetyl and BF$_2$-β-diketonate 2H-pyran-2-ones, and the fourth on conjugated alkenes with a nitro group. The fluorophores were synthesized using organic synthetic methods, including the Suzuki–Miyaura and Heck reactions, the Horner–Wadsworth–Emmons reaction, the Bucherer reaction, Buchwald–Hartwig amination, mixed Claisen condensation, Knoevenagel condensation, cyclization to the 2H-pyran-2-one ring, and the Henry reaction. This enabled the preparation of structurally diverse compound generations, allowing systematic investigation of the influence of the D, π and A on their optical and binding properties. The optical properties of the prepared fluorophores were studied in solvents of different polarity, namely hexane, dichloromethane, acetonitrile, methanol, and HEPES buffer. We showed that absorption and emission maxima, Stokes shifts, and quantum yields are significantly influenced by the nature of the acceptor A and the length and structure of the π-conjugated system. Extension of the π-system and stronger acceptor motifs generally led to a bathochromic shift of absorption and emission, in some cases even into the near-infrared region. An important part of the thesis also involved in vitro evaluation of the optical and binding properties of the prepared fluorophores in the presence of Aβ$_{1-42}$ fibrils. The results showed that these properties are not determined solely by the length of the π-conjugated system, but also by the nature of the aromatic skeleton and by the acceptor and donor groups. Across several generations of compounds, an appropriate combination of these structural elements provided a favorable balance between affinity for Aβ$_{1-42}$ fibrils and the position and intensity of the emission maxima upon binding.

• Keywords: Alzheimer’s disease, amyloid β, D–π–A, fluorophore, 2H-pyran-2-one, nitroalkene, optical properties, binding properties