Preparation of fluorescent tocopherols for use in protein binding and localization with the α-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin α-tocopherol were prepared incorporating fluorophores at the terminus of ω-functionalized 2-n-alkyl-substituted chromanols (1a–d and 4a–d) that match the methylation pattern of α-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein’s binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (α-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4′R,8′R-α-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of α-TTP-mediated inter-membrane transfer of α-tocopherol and to investigate the mechanism of α-TTP function at membranes of different composition.

The aim of this work was to prepare and demonstrate the utility of fluorescent analogues of α-tocopherol as probes for use in protein-binding assays, intracellular localization studies, and transfer assays using vesicles and the tocopherol transfer protein, α-TTP. The NBD-α-tocopherols proved to be excellent ligands of recombinant human α-TTP. One AO-α-tocopherol (n = 4), while still useful, is limited by its very low solubility in buffers.Figure optionsDownload as PowerPoint slide