Razvoj sintezne poti za pripravo derivatov glukozamina kot zaviralcev encima MurA

Budja, Barbara (author), Frlan, Rok (mentor)

Bakterijska odpornost na antibiotike predstavlja vse večjo težavo svetovnemu zdravstvu, saj postajajo številne do zdaj ozdravljive okužbe težje ozdravljive ali celo neozdravljive. Svetovna zdravstvena organizacija (WHO, angl. World Health Organization) jo je označila kot eno od treh največjih groženj javnemu zdravstvu v 21. stoletju. Številne raziskave se zato intenzivno ukvarjajo z odkrivanjem novih protimikrobnih učinkovin, saj se bo lahko človeštvo le na tak način uspešno zoperstavilo temu problemu. Celična stena prokariontske celice je sestavljena iz peptidoglikana, katerega sinteza poteka na treh mestih: v citoplazmi celice, celični membrani ter zunaj celice. Encim MurA ima svoje mesto delovanja v citoplazmi in katalizira prvo reakcijo v sklopu sinteze. Večina antibiotikov, ki so danes na trgu, zavira sintezo peptidoglikana in imajo svoje mesto delovanja zunaj celice v zadnjih stopnjah sinteze. Antibiotikov, ki imajo mesto delovanja znotraj celice, je malo. Eden od teh je fosfomicin, ki zavira delovanje encima MurA. V tej magistrski nalogi smo sintetizirali dva derivata glukozamina. Načrtovali smo ju na podlagi molekulskega modeliranja in s pomočjo posnemanja naravnega substrata. Za osnovni skelet smo uporabili glukozamin, ki nam omogoča pravilno prostorsko usmeritev funkcionalnih skupin in sintezo reverzibilnih zaviralcev, ki bodo sposobni prehajati skozi celično steno in membrane v notranjost bakterije, kjer se nahaja encim MurA. Glavni namen magistrske naloge je bil razvoj sintezne poti za pripravo spojin, ki bi imele na aminoskupini na mestu 2 pripete lipofilne fragmente ter na mestu 6 pripete kisle funkcionalne skupine. Sinteze smo se lotili po dveh poteh. V prvi sintezni poti smo glukozamin zaščitili v obliki benziloksikarbonilnih in acetilnih derivatov, medtem ko smo po drugi sintezni poti uporabili tritilno in benzilno zaščitno skupino. Ugotovili smo, da prva sintezna pot zaradi nizkih izkoristkov (manjši od 10 %) in težav s čiščenjem ni primerna za sintezo končnih spojin, medtem ko je druga sintezna pot primerna za sintezo derivatov na mestu 6, ni pa primerna za sintezo lipofilnih derivatov na mestu 2. Po drugi sintezni poti smo tako pripravili spojino 12, kjer smo hidroksilno skupino oksidirali neposredno do kisline, in spojino 16, pri kateri smo prek tozilata uvedli nitrilno skupino, ki smo jo nato hidrolizirali do kisline. Za obe končni spojini smo v encimskem testu potrdili, da zavirata delovanje encima MurA. Spojini 12 smo določili tudi IC50, ki je bil 133,1 μM.

• Language: Slovenian
• Keywords: odpornost, peptidoglikan, zaviralec, encim MurA, N-acetilglukozamin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-133127
• Publication date in RUL: 12.11.2021

Secondary language

• Language: English
• Title: Development of a synthetic pathway to prepare glucosamine-based MurA enzyme inhibitors

Abstract

Today, antibiotic resistance in bacteria poses increasing problems for the global healthcare system because many curable infections suddenly become less curable or even incurable. WHO catalogs it as one of the top three threats to public healthcare in the 21st century. Much research is done to discover new antimicrobial drugs because that is the only way humanity can address this problem. The cell wall of the procaryotic cell is composed of peptidoglycan. Its biosynthesis takes place in three areas: in the cytoplasm, at the cell membrane, and outside the cell. The enzyme MurA is located in the cytoplasm and catalyzes the first reaction of the pathway. Most of the antibiotics known today inhibit the peptidoglycan synthesis pathway and have their site of action in the final stages outside the cell. There are not many antibiotics with their site of action inside the cell. One of these is fosfomycin which inhibits the activity of the MurA enzyme. In this master’s thesis, we synthesized two glucosamine derivates. We based their design on molecular modeling and mimicking of the natural substrate. We used glucosamine, which allowed us to correctly position the functional groups and synthesize reversible inhibitors capable of passing through the cell wall and membrane into the interior of the bacteria where the MurA enzyme is located. The main objective of this master’s thesis was to develop a synthetic route to prepare compounds in which a lipophilic group is attached to the amino group at C-2 and acidic fragments are attached to the hydroxyl group at C-6. We used two synthetic routes. In the first synthetic route, we protected glucosamine in the form of benzyloxycarbonil and acetyl derivates while in the second route we used trityl and benzyl protecting groups. We found that the first pathway was not suitable for the synthesis of final compounds due to low yield (less than 10%) and purification problems. The second route is suitable for the synthesis of derivates at C-6 but not for the synthesis of lipophilic derivates at C-2. Using the second route, we developed compound 12 where we directly oxidized the hydroxyl group to acid and compound 16 where we added the nitrile group via a tosylate which we then hydrolyzed to acid. Using an enzyme assay, we established that both compounds inhibit the enzyme activity. For compound 12, the IC50 was determined at 133,1 μM.

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