New BODIPY lipid probes for fluorescence studies of membranes

Fluorescent lipid probes have proven to be valuable tools in membrane studies (see Ref. 1 for review). Because the determination of depth-dependent parameters of bilayers can benefit the understanding of membranous structures (2), sets of probes bearing the same fluorophore at different distances from the bilayer surface are potentially quite useful. Ideally, such fluorophores should be apolar enough to localize at the membrane depth that reflects the apolar nature of the surrounding acyl chain region without being strongly influenced by the transbilayer polarity gradient (3). The first probe set designed to achieve this goal was a series of n-(9-anthroyloxy) fatty acids synthesized by Thulborn and Sawyer (4), who showed that the fluorophore resided in the bilayer at a graded series of depths that coincided with the attachment point of the anthroyloxy fluorophore along the acyl chain. Other widely used fluorophores, such as N-dansyl (5) or N-NBD (6, 7), have been shown to have polar characteristics that interfere with localization deep inside the bilayer even when attached to the end of the acyl chain. During the past decade, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore probes have found a wide range of applications in cell biology and biophysics, even though this zwitterionic fluorophore was first synthesized by Treibs and Kreuzer (8) in 1968. The BODIPY fluorophore family has gained popularity because of its overall excellent spectral properties (9, 10), which include high photostability, high molar absorptivities, high quantum yields, and strong, narrow-wavelength emission maxima in the visible region. Although the great majority of BODIPY fluorophores are nearly insensitive to environmental polarity and this property is advantageous for many applications, a few environmentally responsive varieties of BODIPY have been developed (11). Many BODIPY derivatives, including labeled fatty acids and complex lipid probes, have been synthesized (12, 13) and applied in numerous biological studies (1, 14). These probes are commercially available (Invitrogen) (15) but are relatively expensive, presumably because of their rather complicated syntheses. Because the BODIPY group carries no net charge, one might expect it to be a more reliable depth-dependent membrane probe than dansyl or NBD. Despite the seemingly favorable properties, studies of the localization of lipid-attached BODIPY in model membranes have led to conflicting conclusions. Based on iodide quenching studies of different BODIPY probes in phospholipid vesicles and in water, Johnson, Kang, and Haugland (9) concluded that lipid-attached BODIPY fluorophores reside mainly in the bilayer at the expected depth. On the other hand, Menger, Keiper, and Caran (16), using spin-labeled lipids as quenchers incorporated into the membrane, found that BODIPY fluorophores linked to various positions of the phosphatidylcholine acyl chain, including the ω position, reside close to the membrane surface, ∼17−20 A from the bilayer center, as determined by parallax analysis. However, Kaiser and London (17), also using spin-labeled lipid quenchers and parallax analysis, concluded that lipid-attached BODIPY in the bilayer distributes between two populations: in one, the fluorophore is embedded into bilayer along the entire length of the acyl chain; in the other population, BODIPY resides closer to the membrane surface by causing bending/looping of the flexible acyl chain. Even though such dual fluorophore localization and related uncertainties do not diminish the effectiveness of BODIPY for many purposes (e.g., monitoring lipid trafficking within a cell), it can be problematic in other cases, such as in resonance energy transfer experiments in which the fluorophore serves as a molecular ruler, thus requiring its membrane position to be accurately known. The goal of this study was to develop a set of lipid probes, bearing a BODIPY fluorophore at the end of the acyl chain, in which the fluorophore location within the bilayer corresponded to the maximal depth allowed by the acyl chain. Our strategy was to modify the BODIPY structure to counteract the polarity originating from its compensating positive (on nitrogen atom) and negative (on boron atom) internal charges to optimize its capacity for localization in the apolar interior of the bilayer. As a rule, insertion of substituents at positions 1, 3, 5, 7, and 8 of the BODIPY ring (Fig. 1), but not at positions 2 and 6, is known to be well tolerated without negatively affecting emission quantum yield (9). It should be noted that the previously described studies of lipid-bonded BODIPY localization in bilayers (9, 16, 17) were performed using fluorophore containing methyl substituents at positions 5 and 7 and linked to acyl chains via position 3. We reasoned that BODIPY could be optimized for deep bilayer localization by maximizing the number and symmetry of apolar substituents. Our considerations led to the hypothesis that the desired fluorophore should have the BODIPY ring, with identical apolar alkyls at positions 1, 3, 5, and 7, and be linked to acyl chains via position 8. To evaluate this hypothesis, we analyzed the spectral and membrane properties of BODIPY probes with the aforementioned structural features, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8). For this purpose, a novel set of phosphatidylcholines was synthesized with the Me4-BODIPY-8 fluorophore attached to the end of sn-2 fatty acids consisting of three, five, seven, or nine carbon atoms. The location and insertion depth of the Me4-BODIPY-8 fluorophore in unilamellar bilayer vesicles have been assessed by quenching with free iodide (Stern-Volmer analysis) as well as with iodolabeled phosphatidylcholines (parallax analysis) (17). The physical and spectral features of these new phosphatidylcholine probes, in their pure states and in mixtures with other lipids, have been characterized in both bilayer and monolayer model membrane systems to assess the occurrence of lipid-packing distortions caused by the fluorophore as well as concentration-dependent dimerization associated with the BODIPY family of fluorophores (18, 19). Fig. 1 Synthesis of acids XIII–XVI, and structures of 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8), AV12-PC, Me2-BODIPY-3, and iodolabeled probes. AV12-PC, 1-acyl-2-[12-(9-anthryl)-11E-dodecenoyl]-sn-glycero-3-phosphocholine; ...