Synthesis and properties of a highly fluorescent derivative of phosphatidylethanolamine

Abstract

Properties of N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (NBD-PE), prepared by alkylation of the free amino group of phosphatidyl-ethanolamine with 4-chloro-7-nitrobenzofurazan (NBD-C1) are described. The alkylated product containing this fluorochromic group was purified by silicic acid chromatography and had a nitrogen/phosphorus ratio of 3.96 (4.0 theoretical). The NBD-PE was sparingly soluble in distilled water but freely soluble in organic solvents. Both the UV-visible absorption spectrum and the uncorrected fluorescence excitation spectrum of NBD-PE in absolute ethanol showed maxima at approximately 330 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescent intensity and emission maximum wavelength of NBD-PE are strongly dependent on the dielectric constant of the solvent. The fluorescent intensity of NBD-PE in absolute ethanol was directly proportional to its concentration from 1 ng/ml to approximately 3 µg/ml. This compound can be incorporated into phosphatidylcholine–dicetyl phosphate–cholesterol liposomes, rat brain synaptosomal particulate fraction, and human lymphocyte membranes. Incorporation of the derivative into rat liver mitochondrial membranes was facilitated by a postmitochondrial pH 5.1 supernatant fraction. These data indicate that NBD-PE might be a unique and effective probe for the phospholipid regions of membranes.