Sialic acids engineering as a strategy for alternative antibacterial discovery – NEURAPROBE

Objective 1: Design and synthesize reporters of ManNAc and Neu5Ac. Design and synthesize new reporters of Legionaminic, Pseudaminic and 8-epi-Legionaminic acid pathways.
Objective 2: Development of new tools for metabolic cellular imaging Design and synthesize metallic probes for X-fluorescence analysis as an alternative to fluorescent microscopy.
Objective 3: Develop an HTS assay screening to identify inhibitors that may provide new scaffolds for antibacterial/antivirulence drugs.

The synthesis of alkyne and azide derivatives of Neu5Ac and ManNAc has been completed. The metabolic incorporation of these reporters into human cells (HeLA) has been validated. The bioorthogonal ligation methods (CuAAC and SPAAC) were retained. The reporters were detected by confocal fluorescence microscopy after reaction with fluorescent probes.
The synthesis of the ManNAc derivative with an azide in place of the primary alcohol function in position 6 was carried out. This reporter should not become incorporated into mammalian cells unlike bacteria. This hypothesis has been validated in HeLa cells. Experiments are in progress on cultures of E. coli K1 bacteria.
Syntheses of iridium-based probes were carried out. These molecules can be visualized by optical fluorescence but also by X-synchrotron nanoprobes in order to obtain quantitative intracellular information and at the nanoscale. The bioorthogonal ligation reaction with ManNAc chemical reporters has been verified in solution by mass spectrometry and human cell assays are underway to optimize the imaging conditions for reporter incorporation. To improve the solubility of the probes, two versions comprising a PEG spacer were conceptualized and synthesized. The versions without PEG show significant non-specific labeling on HeLa cells. Reaction kinetics studies by HPLC are in progress.
Optimization of the bio-inspired synthetic route from Kdo derivatives has improved the yield and time of this synthetic route for the Kdo-N3 derivative. This optimization also allowed the synthesis of a new derivative of Kdo carrying a thio function, the KdoSSKdo. The metabolic incorporation of this analogue into a bacterial cell (E. coli MG1655) was validated by reaction with a maleimide tool. Obtaining this new analogue will allow double-labeling experiments with different bioorthogonal ligation methods. The synthesis of alkene and alkyne analogs of Leg is underway. The synthesis of analogues of Pse and 8eLeg is also in progress.

The perspectives of the project are to obtain 2D / 3D quantitative information at organelle level on a targeted glycosylation pathway using the bioorthogonal chemical reporter strategy based on metal probes, demonstrating the importance of X-ray fluorescence for a better understanding at a molecular level of the synthesis of oligosaccharides. We have obtained 15 shifts at the ESRF - ID16A-NI (C07).

«Metabolic labeling of bacterial glycans” Guianvarc’h, D ; Boudreux, Y ;Biot, C. ; Vauzeilles, B. Glycoscience 2nd edition, in press.

Sialic acids (SA) are major players in many biological functions such as host-pathogen recognition. Whereas the biosynthesis pathways of SA and the associated enzymes are quite well described in eukaryotes, this is not the case in bacteria.
The objectives of the NEURAPROBE project are to develop new labeling tools and study incorporation of sialic acids using the bioorthogonal chemical reporter strategy, in pathogenic bacteria compared to human cells, as well as well as in cellular infection models, in order to detect key differences and characterize phenotypic variations that will be used to screen chemical libraries.
While the majority of published articles focus on cell-level bioimaging using a fluorescent probe, we will attempt to gain in sensibility and resolution by using metal probes detectable by modern electronic microscopy methods. This engineering has never been used on such biological systems yet.