Development of an Effective Functional Lipid Anchor for Membranes (FLAME) for the Bioorthogonal Modification of the...

The glycosylation of cellular membranes is crucial for the survival and communication of cells. As our target is the engineering of the glycocalyx, we designed a functionalized lipid anchor for the introduction into cellular membranes called Functional Lipid Anchor for MEmbranes (FLAME). Since cholesterol incorporates very effectively into membranes, we developed a twice cholesterol-substituted anchor in a total synthesis by applying protecting group chemistry. We labeled the compound with a fluorescent dye, which allows cell visualization. FLAME was successfully incorporated in the membranes of living human mesenchymal stromal cells (hMSC), acting as a temporary, nontoxic marker. The availability of an azido function─a bioorthogonal reacting group within the compound─enables the convenient coupling of alkyne-functionalized molecules, such as fluorophores or saccharides. After the incorporation of FLAME into the plasma membrane of living hMSC, we were able to successfully couple our molecule with an alkyne-tagged fluorophore via click reaction. This suggests that FLAME is useful for the modification of the membrane surface. Coupling FLAME with a galactosamine derivative yielded FLAME-GalNAc, which was incorporated into U2OS cells as well as in giant unilamellar vesicles (GUVs) and cell-derived giant plasma membrane vesicles (GPMVs). With this, we have shown that FLAME-GalNAc is a useful tool for studying the partitioning in the liquid-ordered (Lo) and the liquid-disordered (Ld) phases. The molecular tool can also be used to analyze the diffusion behavior in the model and the cell membranes by fluorescence correlation spectroscopy (FCS).