20.500.12556/RUL-139718 Poskus sinteze manjšega proteina z uporabo metode na trdnem nosilcu Attempt to synthesize a small protein using solid-phase synthesis SPPS (angl. Solid-Phase Peptide Synthesis, slo. sinteza peptida na trdnem nosilcu) je dokaj enostavna in zanesljiva metoda za sintezo peptidov in manjših proteinov. Ti v naravi predstavljajo zelo pomembne molekule, saj opravljajo mnogo življenjsko pomembnih funkcij. Pri SPPS največkrat uporabljamo Fmoc skupino, kot začasno zaščito na N-koncu modificirane aminokisline. Zaščititi moramo tudi vse ostale funkcionalne skupine aminokislin, ki bi lahko reagirale tekom sinteze, tako da gradimo le eno glavno polipeptidno verigo. Pripravili smo dva peptida – enega manjšega, tako s šaržno sintezo na koloni, s kontinuirnim pretokom in brez trajnih zaščitnih skupin. Po uspešni pripravi krajšega peptida (12 aminokislin) smo se lotili sinteze nekoliko daljšega peptida (26 aminokislin). Po protokolu smo najprej na nosilec vezali prvo modificirano aminokislino, spirali z DMF, odstranili Fmoc skupino s piperidinom v DMF ter zopet spirali. Te cikle smo ponavljali, dokler nismo v verigo vključili vseh predvidenih aminokislin. Peptid smo gradili od C- proti N-koncu. Peptide smo nato očistili na reverzno fazni kromatografski koloni, ter jih analizirali s HPLC in HRMS. SPPS (Solid-Phase Peptide Synthesis) is a fairly simple and reliable method for the synthesis of peptides and smaller proteins. These are very important molecules in nature as they perform many vital functions. The Fmoc group is most commonly used as a temporary protection at the N-terminus of a modified amino acid during SPPS. However, to control the synthesis and to build only one main polypeptide chain, all other functional groups that could react during synthesis also need to be protected. We prepared 2 peptides - one smaller by manual synthesis on a column (a.k.a. batchwise method), as well as with continuous flow and without permanent protecting groups. After successful preparation of the smaller peptide (12 amino acids) we attempted the synthesis of a slightly longer peptide (26 amino acids). Following the established protocol, the first modified amino acid was first coupled to the solid support, washed with DMF, the Fmoc group was removed with piperidine in DMF, and washed again. These cycles were repeated until all the required amino acids were included into the chain. The peptide was constructed from the C- to N-terminus. The peptides were then purified on a reverse-phase chromatographic column and analysed by HPLC and HRMS. Fmoc zaščita aminokisline peptidi kontinuirni pretok SPPS Fmoc protection amino acids peptides continuous flow SPPS true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2022-09-06 13:15:06 2022-09-06 13:15:06 2022-10-21 11:15:32 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 21623 126597635 Renko_Aljaz_-_Poskus_sinteze_manjsega_proteina_z_uporabo_metode_na_trdnem_nosilcu.pdf Renko_Aljaz_-_Poskus_sinteze_manjsega_proteina_z_uporabo_metode_na_trdnem_nosilcu.pdf 1 F16B67483CF97B9940C927A7B1338AEA b3381c9b41e57b91ecfdd8755158d526d3d52efbda4979667ad047dbf4dc8633 1caad186-2dd5-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=160323 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0