Synthesis and evaluation of MurA ligase inhibitors with a glucosamine skeleton

Boj proti bakterijskim okužbam uvrščamo med največje uspehe človeštva, saj je odkritje antibiotikov pripomoglo k izboljšanju kakovosti življenja, hkrati pa je omogočilo tudi hiter razvoj medicine. V zadnjem času smo priča naraščajoči bakterijski odpornost, ki predstavlja vse večjo grožnjo javnemu zdravstvu, zato postaja razvoj novih protibakterijskih učinkovin ključnega pomena. Namen magistrske naloge je bil razvoj sintezne poti za pripravo novih zaviralcev ligaze MurA, ključnega encima, ki katalizira prvo stopnjo biosinteze peptidoglikana in s tem prepreči nadaljnjo sintezo celične stene. V sklopu eksperimentalnega dela smo na podlagi posnemanja naravnega substrata in molekulskega sidranja vnaprej pripravljene knjižnice derivatov glukozamina v aktivno mesto encima MurA načrtovali in sintetizirali nove spojine. Izhajali smo iz N-acetilglukozamina, ki smo mu na dušik na mestu 2 pripenjali različne lipofilne fragmente, mesto 6 pa smo oksidirali do karboksilne kisline. Posebno pozornost smo namenili iskanju najbolj optimalne poti selektivne oksidacije primarne hidroksilne skupine ob prisotnosti nezaščitenih sekundarnih hidroksilnih skupin. Uspelo nam je sintetizirati osem spojin. Sintetiziranim derivatom glukozamina smo na koncu s pomočjo biološkega testiranja na encimu MurA, izoliranem iz bakterije E. coli, z določitvijo vrednosti IC50 in Hillovega koeficienta ovrednotili zaviralno delovanje in specifičnost vezave na tarčni encim. Zmožnost zaviralnega delovanja smo dokazali pri štirih sintetiziranih molekulah, od katerih se je najbolj izkazala spojina 13, saj je njena zaviralna koncentracija primerljiva s spojino 7 in boljša od spojine 10, medtem ko je Hillova konstanta primerljiva s spojino 10, vendar encim zavira pri nižji koncentraciji. Na podlagi dobljenih rezultatov smo zaključili, da je za močnejše in bolj specifično zaviralno delovanje pomembna velikost lipofilnega fragmenta in odsotnost polarnih fragmentov na mestu 2 glukozaminskega skeleta.The fight against bacterial infections is considered one of humanity\u27s greatest successes. The discovery of antibiotics helped to improve the quality of life and the rapid development of medicine. Recently, we have witnessed increasing bacterial resistance, which represents an increasing threat to public health, and the development of new antibacterial agents is becoming crucial. Development of a synthesis route for the preparation of new MurA ligase inhibitors, the key enzyme that catalyses the first stage of peptidoglycan biosynthesis and thus prevents the further synthesis of the cell wall was the purpose of the master\u27s thesis. As part of the experimental work, we planned and synthesized new compounds based on the imitation of the natural substrate and the molecular anchoring of a previously prepared library of glucosamine derivatives into the active site of the MurA enzyme. We started with N acetylglucosamine, to which various lipophilic fragments were attached to the nitrogen at position 2, and position 6, was oxidized to a carboxylic acid. We paid special attention to the search for the most optimal route of selective oxidation of the primary hydroxyl group in the presence of unprotected secondary hydroxyl groups. We managed to synthesize eight compounds. Finally, with the help of biological testing on the MurA enzyme isolated from E. coli, the inhibitory action, and the specificity of binding to the target enzyme were evaluated by determining the IC50 and Hill coefficient of the synthesized glucosamine derivatives. We demonstrated the inhibitory activity of four synthesized molecules, of which compound 13 proved to be the best, as its inhibitory concentration is comparable to compound 7 and better than compound 10, while the Hill constant is comparable to compound 10 but the enzyme inhibits at a lower concentration. Based on the obtained results, we concluded that the size of the lipophilic fragment and the absence of polar fragments at position 2 of the glucosamine skeleton are significant for a tighter and more specific inhibitory action