Sinteza in vrednotenje kislih N-substituiranih derivatov glukozamina kot zaviralcev ligaze MurA

Geč, Katarina (author), Frlan, Rok (mentor)

Boj z bakterijsko odpornostjo na antibiotike postaja v modernem svetu vse večji problem, zato je ključno neprestano prizadevanje za razvoj novih protibakterijskih učinkovin. Z iskanjem zaviralcev encimov, ki sodelujejo pri nastanku bakterijske celične stene, imamo dobro izhodišče, da to uresničimo. Privlačna tarča za sintezo novih učinkovin je encim MurA, ki je s kataliziranjem prvega koraka biosinteze peptidoglikana bistven pri sintezi celične stene v Gram-pozitivnih in Gram-negativnih bakterijah. Namen magistrske naloge je bila sinteza novih potencialnih zaviralcev encima MurA s pripenjanjem kislih funkcionalnih skupin na NH2 skupino derivatov N-acetilglukozamina. Tako smo sintetizirali končne spojine s prosto COOH ali prosto OH skupino substituenta na dušiku na mestu 2. Pri končnih produktih smo želeli imeti v prosti obliki tudi OH skupini na mestih C3 in C4, saj s katalitičnim mestom encima MurA tvorita pomembne interakcije. Sinteze smo izvedli v šestih stopnjah. Najprej smo zaščitili C1-OH skupino s sintezo monometilnega acetala. Nato smo OH skupinama na mestih C4 in C5 uvedli zaščito z benzilidenom. V tretji stopnji je potekla bazična hidroliza acetilne skupine na dušiku, s čimer smo pripravili spojino s prosto aminsko skupino. V naslednjem koraku smo nanjo pripeli različne substituente z zaščiteno kislo skupino. V zadnjih dveh stopnjah pa smo izvedli še odstranitev zaščite kisle skupine substituentov in odstranitev benzilidenske zaščite. Vmesnim in končnim produktom smo z jedrsko magnetno resonanco, infrardečo spektroskopijo, tekočinsko kromatografijo visoke ločljivosti, masno spektrometrijo visoke ločljivosti, tankoplastno kromatografijo, specifično optično sučnostjo in z merjenjem temperature tališča določili čistost in istovetnost. Končnim produktom in spojinam brez benzilidenske zaščite iz predzadnje stopnje sinteze smo izmerili rezidualno aktivnost na ligazi MurA bakterije E. coli. Pri tem je samo spojina 17 izkazala zadostno rezidualno aktivnost (pod 40 %), da smo ji izmerili še IC50. Ta vrednost je znašala 1548 µM in bila previsoka, torej je spojina 17 prešibka pri zaviranju ligaze MurA, lahko pa jo uporabimo za nadaljnji razvoj boljših zaviralcev.

• Language: Slovenian
• Keywords: odpornost, peptidoglikan, N-acetilglukozamin, encim MurA, zaviralci
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-143269
• Publication date in RUL: 10.12.2022

Secondary language

• Language: English
• Title: Synthesis and evaluation of acidic N-substituted glucosamine derivatives as MurA inhibitors

Abstract

The fight against bacterial resistance to antibiotics is becoming an ever-increasing problem in the modern world, making ongoing efforts to develop new antibacterial agents crucial. By finding inhibitors of enzymes involved in bacterial cell wall formation, we are in a good position to achieve this. An attractive target for the synthesis of new compounds is the enzyme MurA, which is essential for cell wall synthesis in Gram-positive and Gram-negative bacteria as it catalyses the first step of peptidoglycan biosynthesis. The aim of this Master thesis was to synthesise new potential inhibitors of MurA by attaching acidic functional groups to the NH2 group of N-acetylglucosamine derivatives. In this way, we synthesised final products with a free COOH or free OH group substituent on the nitrogen on side 2. For the final products, we also wanted the groups OH to be in free form at the C3 and C4 sites, as they form important interactions with the catalytic site of the MurA enzyme. The syntheses were carried out in six steps. First, the C1-OH group was protected by the synthesis of monomethyl acetal. Then the OH groups were protected at the C4 and C5 sites with benzylidene. In the third step, basic hydrolysis of the acetyl group was carried out on the nitrogen to produce a compound with a free amino group. In the next step, various substituents with protected acid group were attached to it. In the last two steps, the acid group protection of the substituents and the benzylidene protection were removed. Purity and identity of the intermediates and final products were determined by nuclear magnetic resonance, infrared spectroscopy, high-performance liquid chromatography, high-performance mass spectrometry, thin-layer chromatography, specific optical rotation and melting point measurements. The residual activity of MurA ligase from E. coli was measured with final products and with benzylidene-free compounds from the penultimate step of the synthesis. Only compound 17 showed sufficient residual activity (below 40 %) to also measure IC50 value. This measured value of 1548 µM was too high, thus compound 17 is too weak at inhibiting MurA ligase but can be used for further development of better inhibitors.

• Keywords: resistance, peptidoglycan, N-acetylglucosamine, MurA enzyme, inhibitors