Razvoj sintezne poti za pripravo N-2 in C-6 substituiranih derivatov glukozamina kot zaviralcev ligaze MurA

Červ, Karla (author), Frlan, Rok (mentor)

Leta 2019 je Svetovna zdravstvena organizacija (ang. World Health Organization) bakterijsko odpornost na antibiotike vključila na seznam desetih največjih groženj svetovnemu zdravstvu. Ker postaja rezistenca na antibiotike vse večji problem, je potreba po načrtovanju in razvoju novih protibakterijskih učinkovin ključna. Med privlačnimi tarčami so encimi, ki so udeleženi v nastanku bakterijske celične stene, natančneje encim UDP-N-acetilglukozamin enolpiruvil transferaza (MurA), ki katalizira prvo stopnjo nastanka peptidoglikana. Namen magistrske naloge je bil razvoj sintezne poti za pripravo N-2 in C-6 substituiranih derivatov glukozamina kot potencialnih zaviralcev ligaze MurA. Z glukozaminom smo posnemali naraven substrat UDP-N-acetilglukozamin (UNAG), s katerim lahko dosežemo pravilno usmerjenost funkcionalnih skupin. Na tak način lahko ustvarimo zaviralce, ki bodo prehajali v bakterije in reverzibilno zavrli encim MurA. Sintetiziranim spojinam smo s tankoplastno kromatografijo, jedrsko magnetno resonanco, masno spektrometrijo visoke ločljivosti, infrardečo spektroskopijo, specifično optično sučnostjo in z merjenjem temperaturnega intervala tališča določili čistost in istovetnost. Izhajali smo iz spojine N-acetilglukozamina, kateri smo najprej zaščitili anomerno hidroksilno skupino (-OH) v obliki metilnega acetala. Nato smo zaščitili -OH skupini na mestih C-4 in C-6 v obliki benziliden acetala. Sledila je še bazična hidroliza amidne vezi, prosto amino skupino pa smo nato zaščitili v obliki benzil karbamata. Odstranili smo benziliden acetalno zaščito in dobili produkt 6, ki je predstavljal našo izhodno spojino za vse nadaljnje reakcije. Spojini smo nato uvedli sililno zaščito na prosto -OH skupino na mestu C-6, -OH skupine na mestu C-3 in C-4 pa smo zaščitili s tvorbo benzoilnega estra. Nato smo odstranili sililno zaščito in izolirali spojino 9. Sledilo je še alkiliranje proste -OH skupine s klorocetno kislino in hidroliza benzoilnih zaščitnih skupin. Nastanka spojin 11 in 12 nismo mogli dokazati, saj je bil izkoristek reakcije prenizek, da bi lahko izolirali in analizirali produkta. V prihodnjih raziskavah bi se bilo smiselno osredotočiti na uvedbo bolj stabilnih zaščitnih skupin, ki bi povečale možnost za nastanek končnega produkta s potencialno sposobnostjo zaviranja.

• Language: Slovenian
• Keywords: peptidoglikan, encim MurA, zaviralec, bakterijska odpornost, derivati glukozamina
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151139
• Publication date in RUL: 30.09.2023

Secondary language

• Language: English
• Title: Development of a synthetic route for the preparation of N-2 and C-6 substituted glucosamine derivatives as MurA ligase inhibitors

Abstract

In 2019, the World Health Organization (WHO) included bacterial resistance to antibiotics in its list of the top ten global public health threats. As antibiotic resistance in bacteria becomes an increasing problem, the need to design and develop new antibacterial agents is critical. Attractive targets include enzymes involved in the formation of the bacterial cell wall, in particular the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the very first step of peptidoglycan synthesis. The aim of this master's thesis was to develop a synthetic route for the preparation of N-2- and C-6-substituted glucosamine derivatives as potential inhibitors of MurA ligase. Glucosamine was used to mimic the natural substrate UDP-N-acetylglucosamine (UNAG) to achieve proper alignment of the functional groups. This approach allows us to produce inhibitors that can enter bacteria and reversibly inhibit the MurA enzyme. The purity and identity of the synthesized compounds were characterized by thin layer chromatography, nuclear magnetic resonance, high resolution mass spectrometry, infrared spectroscopy, specific optical rotation and by measuring the melting point. Starting from N-acetylglucosamine, we first protected the anomeric hydroxyl group (-OH) in the form of a methyl acetal. Then we protected the -OH groups at positions C-4 and C-6 as benzylidene acetals. Next, we performed base hydrolysis of the amide bond, and the free amino group was protected as a benzyl carbamate. This was followed by removal of the benzylidene acetal protection, resulting in the formation of compound 6, which was our starting compound for all subsequent reactions. We then introduced silyl protection for the free -OH group at the C-6 site, while the -OH groups at the C-3 and C-4 sites were protected by the formation of benzoyl esters. We then removed the silyl protection and isolated compound 9. We then performed alkylation of the free -OH group with chloroacetic acid and hydrolyzed the benzoyl protecting groups. However, the formation of compounds 11 and 12 could not be confirmed due to low reaction yields, which prevented isolation and analysis of the products. In future studies, it would be useful to focus on finding more stable protecting groups to increase the possibility of producing the final product with potential inhibitory activity.

• Keywords: peptidoglycan, MurA enzyme, inhibitor, antimicrobial resistance, glucosamine derivatives