doctoral thesis
SYNTHESIS, CHARACTERIZATION AND SELF-ASSEMBLY OF SMALL PEPTIDIC GELATORS BASED ON AMYLOID β- PROTEIN

Tihomir Pospišl (2017)
Sveučilište u Rijeci
Odjel za biotehnologiju
Metadata
TitleSINTEZA, KARAKTERIZACIJA I SAMOUDRUŽIVANJE MALIH PEPTIDNIH GELATORA TEMELJENIH NA PROTEINU AMILOID-β
AuthorTihomir Pospišl
Mentor(s)Leo Frkanec
Abstract
Klasičnim sintetskim metodama peptidne kemije u otopini sintetizirana je serija novih peptidomimetika, tripeptidnih derivata koji u svojoj strukturi sadrţe različite kombinacije aminokiselinskih slijedova (FFA, FAF, AFF) i s različitim zaštitnim grupama na N i C terminalnim završecima. Ispitana su gelirajuća svojstva pripravljenih spojeva u vodi, organskim otapalima i smjesama različitih otapala. Acetilni tripeptidi s aminokiselinskim slijedom FFA (Phe-Phe-Ala) pokazali su se kao dobri gelatori vode i polarnih otapala (6), ali i aromatskih otapala (5). Acetilni derivati s ostalim aminokiselinskim slijedovima (FAF i AFF) pokazali su izrazito slaba gelirajuća svojstva kao i butirilni tripeptidi sa slijedom FFA. Tripeptidi s benziloksikarbonilom (Z) na N terminalnoj strani (28, 32 i 37) su jako efikasni gelatori aromatskih otapala (o-, m- i p-ksilena i tetralina) te dekalina. Morfologija gelskih niti odreĎena je transmisijskom elektronskom mikroskopijom (TEM). Samoorganizacija molekula i supramolekularne interakcije u novim tripeptidnim gelovima proučavane su različitim spektroskopskim metodama (NMR, FTIR, CD) koje su ukazale na postojanje samoudruţivanja molekula, strukture β-nabrane ploče (paralelno ili antiparalelno orijentirane) povezane vodikovim vezama kod 5 i 6 te kod Z-zaštićenih tripeptida (28, 32 i 37). UV-Vis i fluorimetrijskom spektroskopijom te laserskim pretraţnim konfokalnim mikroskopom ispitano je vezanje tripeptida 6 na amiloidne boje (tioflavin T i kongo-crvenilo) u fiziološkim uvjetima. Fluorescencijskom titracijom vodene otopine tripeptida 6 s tioflavinom T dolazi do porasta emisije te boje i formiranja kompleksa stehiometrije 1:1 s konstantom stabilnosti log K = 2,48. Kongo-crvenilo s tripeptidom 6 tvori proziran gel u vodi dok sam spoj 6 stvara mutan hidrogel pri čemu dolazi do značajnijeg porasta emisije fluorescencije spoja kongocrvenilo u gelu u odnosu na otopinu gdje je neznatan porast emisije. Hidrogel kongo-crvenila i tripeptida 6 čine tanke niti promjera 10-15 nm dok TEM samog hidrogela 6 prikazuje prisutnost ravnih traka promjera 50-500 nm što ukazuje na promjenu morfologije gelova. Laserskim pretraţnim konfokalnim mikroskopom pokazano je da se obje amiloidne boje veţu na gelske niti tripeptida 6. Hidrogel tripeptida 6 omogućio je u fiziološkim uvjetima preţivljenje i proliferaciju stanica HEK293T in vitro te se pokazao kao potencijalni biomaterijal za primjenu u tkivnom inţenjerstvu. Dosadašnja istraţivanja su pokazala da bi novosintetizirani hidrogelator 6 mogao posluţiti kao potencijalni minimalistički model agregiranog Aβ-proteina i omogućiti precizan dizajn i razvoj novih efikasnijih molekula inhibitora ili detektora agregiranja
Keywordstripeptides gels Aβ-protein β-sheet amyloid dyes biomaterial HEK293T cells
Parallel title (English)SYNTHESIS, CHARACTERIZATION AND SELF-ASSEMBLY OF SMALL PEPTIDIC GELATORS BASED ON AMYLOID β- PROTEIN
Committee MembersDražen Vikić-Topić (committee chairperson)
Karlo Wittine (committee member)
Sandra Kraljević Pavelić (committee member)
Mladen Žinić (committee member)
GranterSveučilište u Rijeci
Lower level organizational unitsOdjel za biotehnologiju
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
UDK612
APPLIED SCIENCES. MEDICINE. TECHNOLOGY
Physiology
Study programme typeuniversity
Study levelpostgraduate
Study programmeMedicinal chemistry
Academic title abbreviationdr. sc.
Genredoctoral thesis
Language Croatian
Defense date2017
Promoted date2017-04-07
Parallel abstract (English)
Series of tripeptide FFA, FAF and AFF derivatives with different protecting groups was prepared using classical methods of solution-state peptide synthesis. Prepared tripeptides were tested for gelation of water, various organic solvents and mixtures of solvents. Acetyl FFA derivatives exhibited gelation of water, polar solvents (6) and aromatic solvents (5). However, acetyl FAF and AFF derivatives and butyryl FFA derivatives showed poor gelation abilities. Tripeptides with benzyloxicarbonyl protecting group (28, 32 and 37) exhibited strong gelation of aromatic solvents (o-, m- and p-xylene and tetraline) and decaline. Morphology of prepared gels was investigated by Transmission Electron Microscopy (TEM). Organisation in gel assemblies at the molecular and the supramolecular level determined by using spectroscopic methods (NMR, FTIR, CD) pointed towards the β-sheet type of hydrogen bonding selfassociation of tripeptides in gel aggregates. Binding studies of tripeptide 6 with amyloid dyes, Congo Red and Thioflavin T (ThT) were carried out using fluorescence spectroscopy and confocal microscopy. Fluorescence titration of tripeptide 6 aqueous solution bellow its minimal gelation concentration with Thioflavin T showed increase of ThT emission with increased tripeptide concentration and formation of the 1:1 complex with the association constant, log K = 2,48. Congo Red forms transparent hydrogel with a tripeptide 6 and shows increased fluorescence emission compared to aqueous solution of Congo Red. TEM of the tripeptide hydrogel with Congo Red revealed a change in fiber morphology compared to 6 hydrogel. TEM images of the nanofibrous hydrogel network show the presence of a mixture of fibers and straight ribbons with diameters in the range of 50–500 nm while the hydrogel network together with Congo Red contains small fibrils with diameters in range of 10-15 nm. Confocal microscopy revealed that the both dyes bind the hydrogel fibers. As a potential biomaterial, 6 was established as a stable and biocompatible physical support for HEK293T cells in vitro. Tripeptide 6 efficiently supported survival and promoted proliferation of HEK293T cells encapsulated within a three-dimensional nanofiber network. Furthermore, these results suggest the need for further evaluation of in vitro biocompatibility and bioactivity of this tripeptide on neural stem cells upon encapsulation for possible tissue engineering application. Likewise, tripeptide 6 could serve as minimalistic model of aggregated Aβ-protein and enable development of new amyloid aggregation inhibitors.
Parallel keywords (Croatian)tripeptidi gelovi protein amiloid-β β-nabrana ploča amiloidne boje biomaterijal HEK293T stanice
Versionaccepted version
Resource typetext
Access conditionOpen access
Terms of usehttp://creativecommons.org/licenses/by-nc-nd/4.0/
Noteaccepted version
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:864317
CommitterLea Lazzarich