Fluorescence emission spectra of 2a in the presence of liposomes made from POPC (the stronger emission spectrum in figures A, B and C). The weaker emission spectra, due to fluorescence quenching, were obtained using (A) POPC/Tempo PC (85/15, mol/mol), (B) POPC/5-DOXYL PC (85/15, mol/mol), and (C) POPC/12-DOXYL PC (85/15, mol/mol).

In previous studies, we have shown that molecular umbrellas can transport small polar molecules such as glutathione across fluid liposomal membranes.28 Although the precise mechanism of this transport remains to be established, we have postulated one in which the umbrella first approaches a lipid bilayer in a fully exposed conformation (structure A in Scheme 2).25 Hydrophobic interactions with the membrane interior then leads to an adsorbed state in which the hydrophilic faces are in contact with the polar head group region and the hydrophobic faces are in intimate contact with the hydrocarbon region of the lipid bilayer (structure B). Subsequent absorption into the interior of the membrane, being driven by hydrophobic forces, then affords structure C. Translocation to the adjoining leaflet, 180° rotation and reversal of steps B and A (not shown) then releases the conjugate from the other side of the membrane. In essence, we have postulated that the molecular umbrella masks the hydrophilicity of the polar agent as it crosses the hydrocarbon core of the bilayer. Also shown in Scheme 2 are three additional membrane-bound states that are possible. Here, D is analogous to C, except that the molecular umbrella has an inverted orientation, where the scaffold is in intimate contact with the aqueous phase. Structure E is similar to that of D, except that the ligand is now fully immersed in the aqueous phase. Finally, F depicts a state in which the molecular umbrella occupies an intermediate depth within a thinned region of the bilayer. Of these possibilities, only C, D and F represent shielded states of the type that is indicated in Scheme 1, and A and E represent exposed states. The “flattened” structure that is shown as B can viewed as a “hybrid” state, where the pendant ligand if fully exposed to the aqueous phase, but is shielded from neighboring lipids by the amphiphilic walls that are attached to it.

Schemes 3, 4 and 5 show the synthetic approach that we have used in preparing these target compounds. Thus, acylation of the methyl ester of glycine with bromoacetic anhydride to give 4, followed by nucleophilic displacement by pyranine afforded 5 (Scheme 3). Subsequent saponification and conversion to the corresponding triethylammonium salt gave 6 and 7, respectively. The conversion to the triethylammonium salt was found necessary to solubilize this trisulfate in organic solvents. Condensation of 7 with N¹,N³-spermidinebis[cholic acid amide] and N¹,N³-spermidinebis(L-lysine-dicholylamide) yielded conjugates 1a and 2a, respectively (Scheme 4). Persulfation of 1a and 2a then afforded the corresponding sulfate derivatives, 2a and 2b. The synthesis of the non-umbrella analog was carried out in a similar way by first alkylating pyranine with methyl bromoacetate, followed by substitution with methylamine, to give 8 and 3, respectively (Scheme 5).

In this work, we have made extensive use of the parallax method of analysis.29-34 In brief, this technique measures relative fluorescence quenching efficiencies of a series of nitroxide-labeled phospholipids that lie at shallow, medium and deep locations within a lipid bilayer with respect to a membrane-bound fluorophore. Comparison of these quenching efficiencies then allows one to estimate the average depth of the fluorophore. Three nitroxide-labeled phospholipids that have been frequently used for this purpose are shown in Chart 1. A shallow quencher, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine (Tempo PC), has a fluorescence-quenching nitroxide that is positioned in its head group. A medium quencher, 1-palmitoyl-2-stearoyl-(5-DOXYL)-sn-glycero-3-phosphocholine (5-DOXYL PC) has the nitroxide attached to the sn-2 chain just below the head group region. Finally, a deep quencher, 1-palmitoyl-2-stearoyl-(12-DOXYL)-sn-glycero-3-phosphocholine (12-DOXYL PC), has the nitroxide further down the sn-2 chain. Thus, when a membrane-bound fluorophore lies at or near the surface of a phospholipid bilayer, the fluorescence quenching efficiency for Tempo PC is expected to be greater than for that of 5-DOXYL PC and 12-DOXYL PC. In contrast, if the fluorophore is buried within the hydrocarbon interior of the bilayer, then greater fluorescence quenching is expected with 5-DOXYL PC and 12-DOXYL PC. Analysis by use of these three nitroxides allows calculation of the depth of a fluorophore at the 1-2 Å level of resolution.31

With this goal in mind, we have introduced a unique class of compounds that we have termed, “molecular umbrellas”. Such structures are composed of two or more facially amphiphilic units (“walls”) that are covalently attached to a central scaffold. In a sense, these “amphomorphic” compounds mimic the structure and function of umbrellas by being able to cover an attached agent and shield it from an incompatible environment.21-28 Thus, when immersed in a hydrocarbon environment, a molecular umbrella can adopt a shielded conformation in which the facially amphiphilic units not only mask their own hydrophilicity, but also that of the attached agent. When immersed in an aqueous environment, these same molecules can create an exposed conformation that allows each of the hydrophilic faces and the bound hydrophilic agent to make direct contact with water. A stylized illustration of a molecular umbrella in an exposed and a shielded conformation is shown in Scheme 1. Here, the shaded and unshaded rectangles represent hydrophobic and hydrophilic faces of the amphiphilic units, respectively, and the lightly shaded oval represents a covalently attached, hydrophilic agent.

Schemes 3, 4 and 5 show the synthetic approach that we have used in preparing these target compounds. Thus, acylation of the methyl ester of glycine with bromoacetic anhydride to give 4, followed by nucleophilic displacement by pyranine afforded 5 (Scheme 3). Subsequent saponification and conversion to the corresponding triethylammonium salt gave 6 and 7, respectively. The conversion to the triethylammonium salt was found necessary to solubilize this trisulfate in organic solvents. Condensation of 7 with N¹,N³-spermidinebis[cholic acid amide] and N¹,N³-spermidinebis(L-lysine-dicholylamide) yielded conjugates 1a and 2a, respectively (Scheme 4). Persulfation of 1a and 2a then afforded the corresponding sulfate derivatives, 2a and 2b. The synthesis of the non-umbrella analog was carried out in a similar way by first alkylating pyranine with methyl bromoacetate, followed by substitution with methylamine, to give 8 and 3, respectively (Scheme 5).

Schemes 3, 4 and 5 show the synthetic approach that we have used in preparing these target compounds. Thus, acylation of the methyl ester of glycine with bromoacetic anhydride to give 4, followed by nucleophilic displacement by pyranine afforded 5 (Scheme 3). Subsequent saponification and conversion to the corresponding triethylammonium salt gave 6 and 7, respectively. The conversion to the triethylammonium salt was found necessary to solubilize this trisulfate in organic solvents. Condensation of 7 with N¹,N³-spermidinebis[cholic acid amide] and N¹,N³-spermidinebis(L-lysine-dicholylamide) yielded conjugates 1a and 2a, respectively (Scheme 4). Persulfation of 1a and 2a then afforded the corresponding sulfate derivatives, 2a and 2b. The synthesis of the non-umbrella analog was carried out in a similar way by first alkylating pyranine with methyl bromoacetate, followed by substitution with methylamine, to give 8 and 3, respectively (Scheme 5).

In this study, we have chosen Cascade Blue as a “surrogate agent” because of its high polarity and strong fluorescence. Specific conjugates that were selected as synthetic targets were a di-walled and a tetra-walled molecular umbrella having moderate (i.e., hydroxyl-) facial hydrophilicity (i.e., 1a and 2a, respectively), and analogs having strong (i.e., sulfate-) facial hydrophilicity (i.e., 1b and 2b, respectively). In addition, a non-umbrella derivative (3) was chosen as a control compound (Chart 2). To enhance their conformational flexibility, a glycine “handle” was also included in each of the molecular umbrellas.