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Odpornost proti antibiotikom v bakterijah postaja vse bolj resen problem za globalni zdravstveni sistem, saj postajajo številne prej ozdravljive okužbe manj ozdravljive ali celo neozdravljive. Svetovna zdravstvena organizacija (WHO) je bakterijsko odpornost proti antibiotikom uvrstila med tri največje grožnje za javno zdravstvo v 21. stoletju. Razvoj novih protimikrobnih učinkovin tako postaja ena glavnih prioritet, saj se lahko le na ta način izognemo tej težavi. Peptidoglikan je pomemben gradnik bakterijske celične stene, ki se sintetizira v treh različnih delih celice: v citoplazmi, celični membrani in zunaj celice. Prva stopnja reakcijske poti poteka v citoplazmi, kjer encim MurA katalizira pomemben korak. Večina trenutnih protibakterijskih zdravil zavira sintezo peptidoglikana v zadnjih stopnjah sinteze, ki potekajo izven celice. Kljub temu nekaj antibiotikov deluje znotraj celice, med njimi je fosfomicin, ki zavira aktivnost encima MurA v citoplazmi. Ta magistrska naloga se osredotoča na sintezo dveh derivatov glukozamina, ki temeljita na molekularnem modeliranju in posnemanju naravnega substrata. Za pravilno usmerjenost funkcionalnih skupin in sintezo reverzibilnih zaviralcev smo kot osnovno spojino uporabili N-acetilglukozamin, ki lahko prodira skozi celično steno in membrano v notranjost bakterij, kjer se nahaja encim MurA. Glavni cilj je bil razviti sintezno pot za pripravo spojin, v katerih so kisli fragmenti pripeti na hidroksilno skupino na mestu 6. Najprej smo zaščitili vse funkcionalne skupine N-acetilglukozamina, razen hidroksilne skupine na mestu 6. Spojino 11 smo sintetizirali z direktno uvedbo ocetne kisline na hidroksilno skupino, s pomočjo kloroocetne kisline. Pri sintezi spojine 9 pa smo sprva morali hidroksilno skupino aktivirati s tvorbo tozilata, preko katerega smo potem tvorili derivat z dimetil malonatom, ki smo ga na koncu hidrolizirali do karboksilne kisline. Z encimskim testom smo potrdili, da sta obe spojini zavirali encimsko aktivnost MurA, pri čemer je imela spojina 9 vrednost IC50 84 μM in spojina 11 vrednost IC50 14 µM. Antibiotic resistance in bacteria is becoming an increasingly serious problem for the global healthcare system, leading to a reduction in the effectiveness of previously curable infections and even making some infections incurable. The World Health Organization (WHO) has listed it as one of the biggest threats to public healthcare in the 21st century. To address this issue, significant research efforts are being invested into discovery of new antimicrobial drugs. Peptidoglycan is an important component of the bacterial cell wall, and its biosynthesis occurs in three regions: the cytoplasm, the cell membrane, and outside the cell. The MurA enzyme is present in the cytoplasm and initiates the pathway by catalyzing the first reaction. The majority of currently available antibiotics inhibit the peptidoglycan synthesis pathway, with their site of action in the later stages outside the cell. However, a few antibiotics act inside the cell, such as fosfomycin, which inhibits the activity of the MurA enzyme. This master’s thesis focused on synthesizing two glucosamine derivatives based on molecular modeling and mimicking of the natural substrate. N-acetylglucosamine was utilized to correctly align functional groups and develop reversible inhibitors capable of penetrating the cell wall and membrane, reaching the bacteria’s interior where the MurA enzyme is located. The primary purpose of the master’s thesis was to develop a synthetic route for preparing compounds that have acidic fragments attached to the hydroxyl group located at position 6. Firstly, all functional groups of N-acetylglucosamine, except for the hydroxyl group at position 6, were protected. Compound 11 was synthesized by directly introducing acetic acid onto the hydroxyl group using chloroacetic acid. For the synthesis of compound 9, the hydroxyl group was first activated by forming a tosylate, which was then utilized to form a derivative with dimethyl malonate. This derivative was finally hydrolyzed to yield carboxylic acid form. Enzyme testing confirmed that both compounds inhibited enzyme activity, with compound 9 having an IC50 value of 84 μM and compound 11 having an IC50 value of 14 µM.

Zaskrbljujoče povečanje in širjenje mehanizmov odpornosti bakterijskih patogenov je danes eden od največjih svetovnih problemov zdravstvenega sistema, zato je raziskovanje na tem področju nujno. UDP-N-acetilglukozamin enolpiruvil transferaza (MurA) je encim, ki katalizira prvo stopnjo sinteze peptidoglikana. MurA je ključen za rast in preživetje tako grampozitivnih kot tudi gramnegativnih bakterij, ni pa prisoten v evkariontskih celicah, zato je kot tarča zelo zanimiv, hkrati pa je kot tarča validiran s fosfomicinom. Namen magistrske naloge je bila sinteza potencialnih zaviralcev encima MurA iz N-acetilglukozmina z modifikacijami na mestu N-2 in s COOH skupino na mestu C-6. Produkte smo načrtovali na podlagi molekulskega modeliranja in s pomočjo posnemanja naravnega substrata. N-acetilglukozamin smo uporabili zato, ker posnema substrat UDP-GlcNAc. Z njim lahko dosežemo pravilno usmerjenost funkcionalnih skupin in lažje prehajanje skozi celično membrano bakterij. Hidroksilni skupini na mestih C-3 in C-4 pa omogočata pomembne interakcije z aktivnim mestom. Sintezo spojin smo izvedli v šestih stopnjah. Začeli smo z zaščito hidroksilnih skupin na mestih C-1, C-3 in C-4. C-1 hidroksilno skupino smo zaščitili z O-metiliranjem, drugi dve pa s tvorbo benziliden acetala. Pri tretji stopnji smo izvedli bazično hidrolizo amidne vezi. Na prosto aminsko skupino smo v naslednjem koraku pripeli lipofilne fragmente in odščitili benziliden acetalno zaščito s kislinsko katalizirano hidrolizo. V zadnji stopnji smo hidroksilno skupino na mestu C-6 oksidirali do karboksilne kisline. Spojinam iz zadnje in predzadnje stopnje sinteze smo določali rezidualno encimsko aktivnost (RA) na encimu MurA iz bakterije E. Coli. Spojinam, ki so imele RA manjši od 50 % smo določali IC50. Le dve spojini (spojini 6b in 7d) sta pokazali dovolj zaviralnega učinka, da smo jima določili tudi IC50. Spojini sta šibka zaviralca encima MurA z IC50 371 in 375 µM, ki predstavljata potencialno izhodišče za razvoj močnejših in selektivnejših zaviralcev encima MurA. The alarming increase and spread of resistance mechanisms of bacterial pathogens is one of the biggest problems of the global health system today. Therefore, research in this area is of critical importance. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an enzyme that catalyzes the first step of peptidoglycan synthesis. MurA is critical for the growth and survival of both Gram-positive and Gram-negative bacteria, but is not found in eukaryotic cells, making it a very attractive target. The aim of the master thesis was to synthesize potential inhibitors of MurA enzyme from N-acetylglucosmin with modifications at the N-2 position and with a COOH group at the C-6 position. The products were designed based on molecular modeling and by mimicking the natural substrate. N-acetylglucosamine was used because it mimics the substrate UDP-GlcNAc. With it, we can achieve the correct alignment of the functional groups and it passes the bacterial cell membrane more easily. Hydroxyl groups at the C-3 and C-4 positions allow significant interactions with the active site. The synthesis of the compounds was carried out in six steps. We began by protecting the hydroxyl groups at C-1, C-3, and C-4. The C-1 hydroxyl group was protected by O-methylation and the other two by benzylidene acetal formation. In the third step, basic hydrolysis of the amide bond was carried out. In the next step, lipophilic fragments were attached to the free amino group and the benzylidene acetal protection was removed by acid-catalyzed hydrolysis. In the final step, the hydroxyl group at the C-6 position was oxidized to a carboxylic acid. The residual enzyme activity (RA) of the MurA enzyme from the E. coli bacterium was determined for compounds from the last and penultimate synthesis steps. The IC50 was determined for compounds that had a RA of less than 50%. Only two compounds (compounds 6b and 7d) showed sufficient inhibitory activity to determine their IC50. These compounds are weak inhibitors of the MurA enzyme with IC50 of 371 and 375 µM. Nevertheless, they represent a potential starting point for the development of stronger and more selective inhibitors of the MurA enzyme.

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's 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's 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.

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. 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.

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. 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.