New GM1 Ganglioside Derivatives for Selective Single and Double Labelling of the Natural Glycosphingolipid Skeleton

Abstract

Selective single and double labelling of the natural ganglioside GM1 enables one to introduce various markers into different parts of the glycosphingolipid molecule without changing the natural skeleton. To that end, N‐Fmoc‐2‐amino‐, N‐Fmoc‐18‐amino‐ and S‐(ethoxythiocarbonyl)‐18‐mercaptostearic acids have been prepared and coupled with the primary amino group in the sphingosine part of lyso‐GM1 and deAc‐deAcyl‐GM1 gangliosides. The products of these coupling reactions – building blocks 16a, 16b, 16c, 26 and 27 – may be used for the synthesis of GM1 derivatives with one or two fluorescent dye moieties or other labels of various polarities. Examples of various labelling strategies, using hydrophilic and lipophilic photostable fluorescent dyes, have been made available. The GM1 derivatives 17a, 22a and 23c labelled with the fluorescent dye ATTO 647N or the doubly labelled derivative 25b can be used as probes in fluorescence correlation spectroscopy (in conventional microscopy or stimulated emission depletion nanoscopy) to study the diffusion of lipid analogues in model or live cell membranes.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups: Distribution in Phase‐Separated Giant Unilamellar Vesicles

“…We synthesized as et of Gb 3 sphingolipids as depicted in Scheme 1. Altogether eight different glycosphingolipids were synthesized and they consist of the globotriaose head group with two different oligoethylene glycol (PEG) linkers,t o which aB ODIPY fluorophore was attached and the sphingosine.S aturated, unsaturated, a-hydroxylated derivatives, and acombination thereof were prepared, all based on aC 24 fatty acid. C 24 fatty acids were chosen as they are the major constituent (> 50 %) found in natural Gb 3 mixtures such as toxin insensitive erythrocytes, [27] HeLa-cells, [28] and HEp-2 cells.…”

“…To date,o nly af ew examples are found in the literature where synthetic routes towards glycosphingolipids with labeled head groups have been described. [24] Here,wedecided on anew strategy in line with approaches pursued for G M1 and G M3 [25] and focused on head group labeled Gb 3 .The idea is to develop fluorescently labeled Gb 3 glycosphingolipids without altering its binding properties to STxB.W ea ttached af luorophore via an oligoethylene glycol spacer to the 2'-OH group of the middle galactose of the Gb 3 head group,w hich is not involved in STxB binding as deduced from crystal structure analysis [17] and binding studies of different trisaccarides. [26] This approach in turn allows us to alter the fatty acid of the Gb 3 molecules.…”

Labeled gangliosides: their synthesis and use in biological studies

“…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.…”

“…Unfortunately, the brightness and photostability of the NBD fluorophore are rather low, precluding its application in FCS experiments. To mitigate this shortcoming, other lipophilic or hydrophilic fluorophores based on either rhodamine or coumarin structures such as Alexa and ATTO dyes besides the BODIPY fluorophores with greater photostability and quantum yield of fluorescence than NBD have been used to label gangliosides . For metabolic studies, a TMR‐GM1 with the tetramethylrhodamine label linked via a β‐alanine spacer substituting for the natural fatty acyl residue was incorporated into PC12 cells .…”