A dual fluorescent-Raman bioorthogonal probe for specific biosynthetic labeling of intracellular gangliosides.

Abstract

Gangliosides are sialic acid-containing glycosphingolipids integral to the cell membrane, and they are particularly abundant in the nervous system. Aberrant ganglioside metabolism contributes to pathological conditions, including neurodegenerative diseases, lysosomal storage disorders, and cancer. A critical precursor for sialic acid biosynthesis is N-acetyl-D-mannosamine (ManNAc), which can be epimerized from the corresponding UDP-GlcNAc or exogenously supplied through ManNAc derivatives. Currently, tools to visualize and detect gangliosides are very limited and non-specific. Here, we describe a dual fluorescent and Raman-active ManNAlk derivative, phenanthrene-9-Pr₄ManNAlk (MM-JH-2), capable of one-step selective labeling of gangliosides in cells. This modified ManNAlk derivative produces a biologically unique Raman spectral signature, which arises from the carbon-carbon triple bond augmented by conjugation to a fluorescent phenanthrene moiety. Raman maps generated using the alkyne stretching frequency indicate a distribution of MM-JH-2 overlapping with intracellular membrane lipids. Using confocal fluorescence imaging, the cellular transport of labeled gangliosides was tracked. Notably, MM-JH-2 can differentiate between cells that differ in ganglioside biosynthetic flux, such as malignant and nonmalignant cells, as well as distinguish between B cells and T cells. Thus, MM-JH-2 is a next-generation metabolic chemical reporter (MCR) that is Raman-active, fluorescent, and can be broadly applied to cellular studies investigating ganglioside biosynthetic flux.