Bioconjugate Chem. 2002, 13, 351−356
351
Molecular Umbrella-Assisted Transport of Thiolated AMP and ATP Across Phospholipid Bilayers Vaclav Janout, Bingwen Jing, and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015. Received October 9, 2001; Revised Manuscript Received December 4, 2001
Two molecular umbrella-nucleoside conjugates (1a and 1b) have been synthesized via thiolate-disulfide displacement by adenosine 5′-O-(3-thiomonophosphate) and adenosine 5′-O-(3-thiotriphosphate) on an activated dimer derived from cholic acid, spermidine, and 5,5′-dithiobis-(2-nitrobenzoic acid). Both conjugates readily enter the aqueous compartment of liposomes made from 1-palmitoyl-2-oleyol-snglycero-3-phosphocholine (POPC) and release the free nucleoside upon reaction with entrapped glutathione. Approximately 50% of the thiolated form of AMP is released within 20 min at 23 °C; 120 min is required for a similar release of the thiolated form of ATP. The facile cleavage of these conjugates by glutathione, together with the fact that mammalian cells contain millimolar concentrations of this tripeptide in their cytoplasm, suggest that such chemistry may be extended to the practical development of prodrugs, e.g., antisense oligonucleotides that can be delivered into cells.
INTRODUCTION
Lipid bilayers play an essential role in cells by serving as barriers for transport (1). In particular, they permit the selective passage of those molecules and ions that are necessary for maintaining the living state. Due to their hydrophobic core, the ability of many classes of biologically active agents that have therapeutic potential (e.g., nucleosides, opioid pentapeptides, antisense oligonucleotides, and DNA) to enter cells has been limited (2-15). Finding ways to improve the bilayer transport of such molecules is a formidable challenge, and one that has important biomedical implications.
With this challenge in mind, we have introduced a unique class of amphiphilic molecules that interconvert between two different morphological states in response to changes in their microenvironment. These “amphomorphic” compounds, in a sense, mimic the structure and function of umbrellas by being able to cover an attached agent and shield it from an incompatible environment (16, 17). Thus, when two or more facially amphiphilic units (i.e., rigid hydrocarbon units that maintain a hydrophobic and a hydrophilic face) are coupled to a suitable scaffold that contains a hydrophilic agent, immersion of such a molecule in an aqueous environment favors an exposed (or fully exposed) conformation, where hydrophobic interactions are maximized and where the hydrophilic faces are hydrated. When immersed in an hydrocarbon environment (e.g., the interior of a lipid bilayer), the umbrella then favors a shielded conforma* To whom correspondence should be addressed. E-mail:
[email protected].
tion such that intramolecular dipole-dipole and hydrogen bonding interactions are maximized, and the hydrophobic faces are solvated. A stylized illustration of a molecular umbrella in shielded, exposed, and fully exposed conformations is presented in Scheme 1. Here, each amphiphilic unit appears as a doubly shaded rectangle having a hydrophobic (darkened) and a hydrophilic (lightly shaded) face; the lightly shaded oval corresponds to a hydrophilic agent. Our working hypothesis has been that molecular umbrella conjugates can cross lipid bilayers through the following sequence of events: (i) diffusion to the membrane surface in an exposed or fully exposed state, (ii) insertion into the outer monolayer leaflet by flipping into a shielded state, (iii) diffusion to the inner monolayer leaflet, and (iv) entry into the adjoining aqueous phase via the reversal of steps ii and i (Scheme 2). In recent studies, we have obtained compelling evidence for an “umbrella” mechanism of permeation for the transport of an umbrella conjugate of glutathione (γ-GluCys-Gly, GSH) across phospholipid bilayers (18). Specifically, we found that the ability of this conjugate to cross these bilayers depended more on their facially amphiphilicity than on their hydrophobic/hydrophilic balance. Thus, the permeation of molecular umbrellas across lipid membranes takes place by a mechanism that is different from classic solution-diffusion pathways, where permeation rates are controlled by the permeant’s solubility and diffusivity in the hydrocarbon region of the membrane (3). In an effort to expand the scope of molecular umbrellas as membrane transporters, we have begun to turn our attention toward the bilayer transport of antisense oligonucleotides. As a first step in this direction, we sought to demonstrate the feasibility of transporting cleavable forms of adenosine 5′-monophosphate (AMP) and adenosine 5′-triphosphate (ATP) across phospholipid bilayers. Although several studies have been aimed at creating prodrugs of nucleoside 5′monophosphates, reports of membrane transporters of nucleoside 5′-triphosphates are rare. In one recent study, where it was
10.1021/bc015564m CCC: $22.00 © 2002 American Chemical Society Published on Web 01/30/2002
352 Bioconjugate Chem., Vol. 13, No. 2, 2002
Janout et al.
Scheme 1
Scheme 2
claimed that a cholesteryloxycarbonyl-ATP conjugate releases of free ATP into the aqueous compartment of liposomes, the transport activity that was observed was only modest; ca. 10% after 9 days at an unspecified temperature (19). In this paper, we describe the synthesis of molecular umbrella conjugates of thiolated forms of AMP and ATP. We also show that these conjugates deliver the free nucleosides into liposomes, made from 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC), with half-lives that are less than 20 and 120 min at 23 °C for the monophosphate and triphosphate, respectively. MATERIALS AND METHODS
General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. 1-Palmitoyl-2-oleoyl-sn-glycero-phosphocholine was obtained from Avanti Polar Lipids (Alabaster, AL). Adenosine 5′-O-(3-thiotriphosphate) and adenosine 5′-O-(3-thiomonophosphate) were purchased from Fluka and used directly. 5,5′-Dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) was purchased from Aldrich (Milwaukee, WI) and used as obtained. All dialysis experiments were made using a 1.5-mL equilibrium dialysis cell (Fischer Scientific) and SpectraPor #7 dialysis tubing (MWCO 50000). All 1H NMR spectra were recorded on a Bruker 360 MHz instrument; chemical
shifts are reported in ppm and are referenced to residual solvent. The buffer that was used in all experiments was composed of 0.1 M H3BO3 and 2 mM EDTA, where the pH was adjusted to 7.0 by use of 1 M NaOH. All UV spectra were recorded using a Cary 300 Bio UV/VIS spectrophotometer. Molecular Umbrella-ATP Conjugate 1b. To a solution of 12.6 mg (5.7 µmol) of 6 in a mixture of 400 µL of CH3OH and 150 µL of H2O was added a solution that was made from 3.12 mg (5.7 µmol) of the tetralithium salt of adenosine 5′-O-(3-thiotriphosphate) and 150 µL of water. A homogeneous orange-colored solution was produced, and the solution was stirred for a total of 72 h at room temperature. Concentration under reduced pressure, followed by preparative thin-layer chromatography (silica gel, CHCl3/CH3OH/H2O, 60/40/10, v/v/v), afforded 2.51 mg (24.9%) of the desired conjugate, 1b, having Rf 0.45 and 1H NMR (CD3OD) δ 842 (d, 1 H), 8.19 (s, 1 H), 8.06 (m, 1 H), 7.70 (m, 2 H), 6.05 (m, 1 H), 4.67 (m, 1 H), 4.45 (m, 1 H), 4.25 (m, 3 H), 3.92 (m, 2 H), 3.78 (s, 2 H), 3.35 (m, 6 H), 3.00 (m, 4 H), 0.89-2.20 (m, 66 H), 0.68 (d, 6 H). HRMS for C72H110N9O24P3S2Na3 (MNa+) Calcd: 1710.6006. Found: 1710.6094. Molecular Umbrella-AMP Conjugate (1a). Using procedures similar to those used for the synthesis of 1b, the molecular umbrella-AMP conjugate (1a) was ob-
Transport across Phospholipid Bilayers
tained in a 53% isolated yield, having Rf 0.41 (silica gel, CHCl3/CH3OH/H2O, 65/25/4, v/v/v) and 1H NMR (CD3OD) δ 841 (d, 1 H), 8.19 (s, 1 H), 8.03 (m, 1 H), 7.68 (m, 2 H), 6.06 (m, 1 H), 4.66 (m, 1 H), 4.30 (m, 1 H), 4.20 (m, 3 H), 3.92 (m, 2 H), 3.78 (s, 2 H), 3.35 (m, 6 H), 3.00 (m, 4 H), 0.90-2.25 (m, 66 H), 0.69 (d, 6 H). HRMS for C72H109N9O17PS2Na2 (MNa+) Calcd: 1512.6910. Found: 1512.6959. Vesicles for Transport Experiments. Typically, 2.0 mL of a 20 mg/mL solution of POPC in chloroform were transferred to a Pyrex test tube, and the solvent was evaporated by rotating the tube under a stream of nitrogen, resulting in a thin lipid film. The last traces of solvent were removed under reduced pressure (25 °C, 72 h,
