Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.
Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-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 (alpha-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-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Ont., Canada L2S 3A1.
Phillip
P
Nava
Matt
M
Cecchini
Sara
S
Chirico
Heather
H
Gordon
Samantha
S
Morley
Danny
D
Manor
Jeffrey
J
Atkinson
D001667:Binding, Competitive / Q000187:drug effects; D002352:Carrier Proteins / Q000187:drug effects*; D005453:Fluorescence; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000187:drug effects; D013329:Structure-Activity Relationship; D024505:Tocopherols / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D014961:X-Ray Diffraction
D002352:Carrier Proteins; D011994:Recombinant Proteins; C017763:alpha-tocopherol transfer protein; D024505:Tocopherols