Superoxide organic chemistry within the liposomal bilayer, part II: a correlation between location and chemistry

Coumarin ester derivatives 1, substituted at C-4 and/or C-12 with alkyl chains, were synthesized and intercalated within DMPC liposomal bilayers. By correlating the 13C chemical shift with medium polarity [ET(30)], the relative location of these substrates within the liposomal bilayer was determined. The length of the alkyl chain substituents clearly influences the lipophilicity of the substrates and their location and orientation within the liposome: Superoxide readily saponifies the C-12 esteric linkage of 1, when this reaction site lies in a polar region of the liposome (ET(30) > 45 kcal/mol), to give the corresponding 7-hydroxy coumarin derivatives 2. However, when C-12 lies deeper and is hence less available to O2√−, the lactonic carbon C-2, which lies in a shallower region (ET(30) = 43–49), is the preferred site for superoxide-mediated cleavage. When coumarin 1 is disubstituted with long chains at both C-12 and C-4, these derivatives lie deep within the bilayer and react only slowly with O2√−. These results indicate there is indeed a correlation between location within the bilayer and substrate reactivity. Contrary to the suggestion of Dix and Aikens (Chem. Res. Toxicol.6:2–18; 1993) superoxide can penetrate deep within the liposomal bilayer. Nevertheless, its concentration drops precipitously (to not, vert, similar16% of what it is near the interface) below ET values of 38, thereby precluding substantial reaction with many highly lipophilic substrates. This work also confirms the findings of others that reactions of small oxy-radicals occur within cellular membranes and appear to be of significant biological importance.