The emergence of antibiotic resistance has prompted active research in the development of antibiotics with new modes of action. Among all essential bacterial proteins, transglycosylase polymerizes lipid II into peptidoglycan and is one of the most favorable targets because of its vital role in peptidoglycan synthesis. Described in this study is a practical enzymatic method for the synthesis of lipid II, coupled with cofactor regeneration, to give the product in a 50-70% yield. This development depends on two key steps: the overexpression of MraY for the synthesis of lipid I and the use of undecaprenol kinase for the preparation of polyprenol phosphates. This method was further applied to the synthesis of lipid II analogues. It was found that MraY and undecaprenol kinase can accept a wide range of lipids containing various lengths and configurations. The activity of lipid II analogues for bacterial transglycolase was also evaluated.
The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.
We herein describe the first synthesis of iminosugar C-glycosides of α-d-GlcNAc-1-phosphate in 10 steps starting from unprotected d-GlcNAc. A diastereoselective intramolecular iodoamination–cyclization as the key step was employed to construct the central piperidine ring of the iminosugar and the C-glycosidic structure of α-d-GlcNAc. Finally, the iminosugar phosphonate and its elongated phosphate analogue were accessed. These phosphorus-containing iminosugars were coupled efficiently with lipophilic monophosphates to give lipid-linked pyrophosphate derivatives, which are lipid II mimetics endowed with potent inhibitory properties toward bacterial transglycosylases (TGase).
The emergence of antibiotic resistance has prompted active research in the development of antibiotics with new modes of action. Among all essential bacterial proteins, transglycosylase polymerizes lipid II into peptidoglycan and is one of the most favorable targets because of its vital role in peptidoglycan synthesis. Described in this study is a practical enzymatic method for the synthesis of lipid II, coupled with cofactor regeneration, to give the product in a 50–70 % yield. This development depends on two key steps: the overexpression of MraY for the synthesis of lipid I and the use of undecaprenol kinase for the preparation of polyprenol phosphates. This method was further applied to the synthesis of lipid II analogues. It was found that MraY and undecaprenol kinase can accept a wide range of lipids containing various lengths and configurations. The activity of lipid II analogues for bacterial transglycolase was also evaluated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.