Synthesis of Fluorescent Gangliosides

Abstract

Labeled gangliosides are invaluable tools to study their transport and metabolism within cells as well as to determine their distribution in membranes, and their interaction with membrane lipids and proteins. Here I describe established procedures to synthesize ganglioside derivatives with a fluorescent tag either attached to its sialooligosaccharide or ceramide portion. These procedures are chosen as to minimize the integrity of the ganglioside molecule and hence, to leave their native skeleton formally intact. The α-position of the stearic acid residue is favorable for the attachment both of hydrophilic and of lipophilic dyes. In some other cases, and starting from lyso-gangliosides, procedures are described by which a fluorescent tag bound to a short acyl chain replaces the native acyl chain of gangliosides.

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

“…For this purpose we used two astrocytoma cell lines derived from wild-type mouse brain (11+/+C1) and ASA-knockout mouse brain (17−/−A1), respectively ( 13 ). To circumvent the low photostability of NBD, making long-term in vivo imaging difficult, sulfatide carrying an N-linked fatty acid with a more stable BODIPY 500/510 fluorophore (green) was synthesized ( 14 ). Cells were costained with the Golgi marker BODIPY-TR-ceramide (red).…”

“…Then, the cells were washed and BODIPY 500/510-labeled sulfatide was loaded by translipofection as described before. The labeled sulfatide was synthesized by standard methods from lyso-sulfatide and BODIPY 500/510 C12-fatty acid (Thermo Fisher Scientific) ( 14 ). Fluorescence was monitored in an Axiovert 200M inverted microscope equipped with appropriate fluorescence filters, an AxioCam MRc camera in monochrome mode and the AxioVision 4.8.2. software (all from Carl Zeiss).…”

Labeled gangliosides: their synthesis and use in biological studies

“…This reacylation can be done with an activated labeled fatty acid or with an activated fatty acid bearing a functional group that subsequently can be modified with a radioactive tag. For the preparation of the necessary lysogangliosides, four procedures have been developed over the last decades and recently described in detail . To this end, gangliosides GM3, GM2, and GM1 have been prepared with a [1‐ 14 C]stearoyl chain in the ceramide moiety and used in enzymatic degradation of gangliosides .…”

“…For the preparation of labeled gangliosides having the appropriate fluorophore in their ceramide, the usual procedure is to reacylate lysogangliosides. This reacylation can be done with an activated fluorescent fatty acid or with an activated fatty acid bearing a protected functional group at the alpha or omega position that subsequently can be unmasked and modified with a desired tag .…”

“…Also, this position of the NBD group was found to leave the binding of the modified lipid to both the activator protein and the degrading enzyme unaffected . This tag position was recently used to decorate gangliosides with various fluorescent dyes . Unfortunately, the brightness and photostability of the NBD fluorophore are rather low, precluding its application in FCS experiments.…”