Submitted on October 18, 2006
Accepted on April 6, 2007
New BODIPY lipid probes for fluorescence studies of membranes
Ivan A. Boldyrev, Xiuhong Zhai, Maureen M. Momsen, Howard L. Brockman, Rhoderick E. Brown, and Julian G. Molotkovsky
Laboratory of Lipid Chemistry, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997
Corresponding Author: jgmol{at}ibch.ru
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 (MeB4B-BODIPY-8) at the end of CB3B-, CB5B-, CB7B- or CB9B-acyls. 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 MeB4B-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of MeB4B-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of MeB4B-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation below 10 mole% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed 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 absence of the 620-630 nm peak associated with BODPY dimer fluorescence and presence of a 570 nm emission shoulder at high MeB4B-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.
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