Solid phase synthesis of cyclic peptides containing muramic acid

U okviru ove disertacije sintetizirana je knjižnica cikličkih peptida i njihovih linearnih analoga s muraminskom kiselinom (Mur) na različitim položajima u odnosu na biološki aktivnu sekvencu Leu-Ser-Lys-Leu (LSKL). Uzimajući u obzir da je muraminska kiselina dipeptidni izoster priprava ortogonalno Fmoc-zaštićene Mur, kao građevne jedinice za sintezu analoga prirodnih produkata na čvrstom nosaču, od posebne je praktične važnosti. Sinteza lineranih glikopeptida provedena je na čvrstom nosaču korištenjem Fmoc-strategije uz reagense O-(benzotriazol-1-il)-1,1,3,3-tetrametilamonijev heksaflorofosfat (HBTU)/1-hidroksibenzotriazol (HOBt) za standardne aminokiseline, te O-(7-azabenzotriazol-1-il)-1,1,3,3-tetrametilamonijev heksafluorofosfat (HATU)/HOBt za ugradnju Mur, na instrumentu za automatsku sintezu peptida. Ciklički peptidi pripravljeni su tzv. glava-rep ciklizacijom linearnih peptida ručnom sintezom na čvrstom nosaču uz reagense HATU ili benzotriaz-1-il-oksitripirolidinfosfonijev heksafluorofosfat (PyBOP). Uklanjanjem peptida s čvrstog nosača dobiveni su glikopeptidi s benzilnom zaštitom na hidroksilnoj skupini C-1 položaja Mur, a nakon katalitičke hidrogenolize dobiveno je 6 linearnih heptapeptida, koji sadrže Asn (važan za vezanje na čvrsti nosač), sekvencu LSKL, te muraminsku kiselinu na različitim položajima (1, 2, 5, 6) koje izmjenjuje s Lys. Deprotekcijom je dobiveno i 6 cikličkih nezaštićenih heptapeptida u kojima je ciklizacija ostvarena preko karboksilne skupine Asn i amino skupine Mur, Lys (bočnog lanca) ili Leu. Provedena su biološka ispitivanja antiproliferativne aktivnosti sintetiziranih spojeva na humanim staničnim linijama karcinoma, antimikrobna aktivnost te interakcija s DNA.In frame of this dissertation the peptide libraries of cyclic peptide and their linear analogs containing muramic acid (Mur) in different positions, according to biologically active sequence Leu-Ser-Lys-Leu (LSKL), were synthesized. Taking into consideration that muramic acid is a dipeptide isoster, preparation of orthogonally Fmoc-protected Mur as a building block for solid phase synthesis of natural products is of particular practical importance. Synthesis of linear glycopetpide was obtained using Fmoc-strategy solid phase peptide synthesis with O-(benzotriazol-1-yl)-1,1,3,3-tetramethylamonium hexafluorophosphat (HBTU)/1-benzotriazole (HOBt) reagents for standard amino acids and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylamonium hexafluorophosphate (HATU)/HOBt for incorporation of Mur, on peptide synthesizer. Cyclic peptides were prepared according to the so called head-to tail cyclisation of linear peptides using manual synthesis on solid phase with HATU or benzotriaz-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) reagents. Cleavage of the peptide from solid phase gave glycopetides with benzyl protected hydroxyl group at C-1 position of Mur, which was followed by catalytic hydrogenolysis to give 6 linear heptapeptides which contain Asn (important for anchoring to resin), sequence LSKL, muramic acid on different positions (1, 2, 5, 6) which alternates with Lys. Deprotection gave 6 cyclic unprotected heptapeptides wherein the cyclization was accomplished via a carboxyl group of Asn and an amino group of Mur, Lys (side chain) or Leu. The effects on proliferation of different human tumor cell lines, antimicrobial activity and interaction with DNA of prepared compounds were investigated