New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a- diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7- tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY-8) at the end of C₃-, C₅-, C₇-, or C₉-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me ₄-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me₄-BODIPY-8 fluorescence quenching by phosphatidyl-cholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me₄-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and ∼506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me₄-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.