Characterization of a Homologous Series of Fluorescent Fatty Amines. Photophysics, Aqueous Solubility and Binding to Albumin and to POPC Bilayers

Abstract

The 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) is a small and very polar group whose fluorescence quantum yield strongly depends on the polarity of its environment. When bound to the polar headgroup of lipid molecules it allows the characterization of the structure and dynamics of lipid bilayers. The solvent dependence of its fluorescence has been exploited by this research group to characterize the kinetics and thermodynamics of the interaction of different amphiphiles with lipid bilayers and proteins.In this work we report on the synthesis and characterization of a homologous series of fluorescent fatty amines (NBD-Cn; n=4, 6, 8, 10, 12, 14 and 16). At 25°C and pH=7.4, the critical aggregation concentration (CAC) in aqueous media range from 2x10−4 M for NBD-C4 to 4x10−9 M for NBD-C12. The partition coefficient to lipid bilayers prepared from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) was also measured for the amphiphiles with a CAC>20 nM (NBD-C4 to NBD-C10) and ranged from 9.5x102 to 3.6x105, with a ΔΔG=-4.9±0.5 kJ/mol per ethyl group. The amphiphiles interacted efficiently with Bovine Serum Albumin (KB=1.7x104 and 7.9x106 M−1 for NBD-C4 and NBD-C10 respectively) and this was inhibited by fatty acids indicating that binding occurs essentially in the same binding site.Some photophysical properties of the amphiphiles in POPC bilayers were also measured and we found no significant variation along the series indicating that the NBD group is located in a region with the same properties regardless of the length of the non-polar group. An exception was noted for the case of NBD-C14 that showed somewhat smaller fluorescence anisotropy. The amphiphiles lifetime decay observed was mono-exponential in water or methanol but when inserted in POPC bilayers a bi-exponential law was required.