New fluorescent cholesterol analogs as membrane probes

New fluorescent cholesterol analogs, (22E,20R)-3 beta-hydroxy-23-(9-anthryl )-24-norchola-5,22-diene (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synth esized, their spectral and membrane properties were characterized. The prob es bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the chole sterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, t he force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N -stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures belo w film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molec ular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fract ion of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lam bda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or li quid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and d ecreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which sh ow also, in accordance with monolayer data, that S-AV-Ch is better accommod ated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle produ ction. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that o f R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV -labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies. (C) 1999 Elsevier Science B.V. All rights reserved.