Locating intercalants within lipid bilayers using fluorescence quenching by bromophospholipids and iodophospholipids.

In previous work, we have been able to determine the depth of intercalated molecules within the lipid bilayer using the solvent polarity sensitivity of three spectroscopic techniques: the 13C NMR chemical shift (δ); the fluorescence emission wavelength (λem), and the ESR ß-H splitting constants (aß-H). In the present paper, we use the quenching by a heavy atom (Br or I), situated at a known location along a phospholipid chain, as a probe of the location of a fluorescent moiety. We have synthesized various phospholipids with bromine (or iodine) atoms substituted at various locations along the lipid chain. The latter halolipids were intercalated in turn with various fluorophores into DMPC liposomes, biomembranes and erythrocyte ghosts. The most effective fluorescence quenching occurs when the heavy atom location corresponds to that of the fluorophore. The results show that generally speaking the fluorophore intercalates the same depth independent of which lipid bilayer is used. KBr (or KI) is the most effective quencher when the fluorophore resides in or at the aqueous phase. Presumably because of iodine's larger radius and spin coupling constant, the iodine analogs are far less discriminating in the depth range it quenches.

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer. Part IV: studies on ketophospholipids.

In our companion paper, we described the preparation and intercalation of two homologous series of dicarbonyl compounds, methyl n-oxooctadecanoates and the corresponding n-oxooctadecanoic acids (n=4-16), into DMPC liposomes. (13)C NMR chemical shift of the various carbonyls was analyzed using an E(T)(30) solvent polarity-chemical shift correlation table and the corresponding calculated penetration depth (in Å). An iterative best fit analysis of the data points revealed an exponential correlation between E(T)(30) micropolarity and the penetration depth (in Å) into the liposomal bilayer. However, this study is still incomplete, since the plot lacks data points in the important area of moderately polarity, i.e., in the E(T)(30) range of 51-45.5 kcal/mol. To correct this lacuna, a family of ketophospholipids was prepared in which the above n-oxooctadecanoic acids were attached to the sn-2 position of a phosphatidylcholine with a palmitic acid chain at sn-1. To assist in assignment and detection several derivatives were prepared (13)C-enriched in both carbonyls. The various homologs were intercalated into DMPC liposomes and give points specifically in the missing area of the previous polarity-penetration correlation graph. Interestingly, the calculated exponential relationship of the complete graph was essentially the same as that calculated in the companion paper based on the methyl n-oxooctadecanoates and the corresponding n-oxooctadecanoic acids alone. The polarity at the midplane of such DMPC systems is ca. 33 kcal/mol and is not expected to change very much if we extend the lipid chains. This paper concludes with a chemical ruler that maps the changing polarity experienced by an intercalant as it penetrates the liposomal bilayer.

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer: Part III: studies on keto esters and acids.

The development of "molecular rulers" would allow one to quantitatively locate the penetration depth of intercalants within lipid bilayers. To this end, an attempt was made to correlate the (13)C NMR chemical shift of polarizable "reporter" carbons (e.g., carbonyls) of intercalants within DMPC liposomal bilayers - with the polarity it experiences, and with its Angstrom distance from the interface. This requires families of molecules with two "reporter carbons" separated by a known distance, residing at various depths/polarities within the bilayer. For this purpose, two homologous series of dicarbonyl compounds, methyl n-oxooctadecanoates and the corresponding n-oxooctadecanoic acids (n=4-16), were synthesized. To assist in assignment and detection several homologs in each system were prepared (13)C-enriched in both carbonyls. Within each family, the number of carbons and functional groups remains the same, with the only difference being the location of the second ketone carbonyl along the fatty acid chain. Surprisingly, the head groups within each family are not anchored near the lipid-water interface, nor are they even all located at the same depth. Nevertheless, using an iterative best fit analysis of the data points enables one to obtain an exponential curve. The latter gives substantial insight into the correlation between polarity (measured in terms of the Reichardt polarity parameter, ET(30)) and penetration depth into the liposomal bilayer. Still missing from this curve are data points in the moderate polarity range.