Title SINTEZA, KARAKTERIZACIJA I SAMOUDRUŽIVANJE MALIH PEPTIDNIH GELATORA TEMELJENIH NA PROTEINU AMILOID-β Title (english) SYNTHESIS, CHARACTERIZATION AND SELF-ASSEMBLY OF SMALL PEPTIDIC GELATORS BASED ON AMYLOID β-
PROTEIN Author Tihomir Pospišil Mentor Leo Frkanec (mentor)Committee member Dražen Vikić-Topić (predsjednik povjerenstva)Committee member Karlo Wittine (član povjerenstva)Committee member Sandra Kraljević Pavelić (član povjerenstva)Committee member Mladen Žinić (član povjerenstva) Granter University of Rijeka Defense date and country 2017, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 612 - Physiology 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
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.
Keywords
tripeptidi
gelovi
protein amiloid-β
β-nabrana ploča
amiloidne boje
biomaterijal
HEK293T stanice
Keywords (english)
tripeptides
gels
Aβ-protein
β-sheet
amyloid dyes
biomaterial
HEK293T cells
Language croatian URN:NBN urn:nbn:hr:193:864317 Promotion 2017-04-07 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2017-04-12 08:34:14
