Bioconjugate Chem. 2003, 14, 1191−1196
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Fully Detachable Molecular Umbrellas as Peptide Delivery Agents Bingwen Jing, Vaclav Janout, and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015. Received May 7, 2003; Revised Manuscript Received October 8, 2003
A persulfated molecular umbrella, derived from cholic acid and spermidine, has been covalently attached to H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) by use of an o-dithiobenzyl carbamate linkage. Treatment of the resulting conjugate (1) with glutathione in solution resulted in the liberation of the free form of the peptide. Addition of 1 to glutathione-entrapped liposomes, prepared from 1-palmitoyl2-oleyol-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and cholesterol [POPC/POPG/cholesterol, 72/4/24 (mol/mol/mol)], resulted in the delivery of DADLE into their aqueous interior.
INTRODUCTION
Scheme 1
Molecular umbrellas are a unique class of amphiphiles that are capable of shielding an attached agent from an incompatible environment. Typically, such molecules are composed of a central scaffold that contains two or more facially amphiphilic units (1). When a hydrophilic agent is attached to an umbrella molecule, immersion in water favors an exposed conformation such that intramolecular hydrophobic interactions are maximized (2). Alternatively, when immersed in a hydrophobic environment, the molecular umbrella can shield the agent by providing a hydrophobic exterior (2). A stylized illustration of shielded and exposed conformers is shown in Scheme 1. Here, the shaded and unshaded rectangles represent hydrophobic and hydrophilic faces, respectively. Recent mechanistic studies have provided strong evidence that facial amphiphilicity plays a major role in umbrella transport across lipid bilayers (3). The fact that facial amphiphilicity is more important than the hydrophobic/hydrophilic balance of such molecules, in promoting bilayer transport, lends strong support for an umbrella mechanism; that is, a permeation pathway involving monolayer insertion of a shielded conformer, transbilayer diffusion and entry to the opposite membrane/water interface. Previous molecular umbrellas that we have designed for peptide transport have employed a 2-nitrobenzoyl-5dithio moiety as a detachable handle (3). When these conjugates enter the aqueous interior of a liposome that contains glutathione (GSH), the peptide is released by means of a thiolate-disulfide exchange reaction (Scheme 2) (4). The fact that mammalian cells contain millimolar concentrations of GSH within their cytoplasm adds relevance to such a strategy from a prodrug standpoint. Two distinct limitations associated with this approach, however, are the need of a cysteine residue for umbrella attachment/detachment, and the peptide is released in a non-native state; that is, as a glutathione conjugate. In this paper, we introduce a new design strategy for the synthesis of molecular umbrella-peptide conjugates that (i) takes advantage of GSH-based thiolate-disulfide interchange reactions, (ii) circumvents the need for a * To whom correspondence should be addressed. E-mail:
[email protected].
Scheme 2
cysteine residue for attachment of the peptide to the umbrella, and (iii) allows for the release of the native form of the peptide. EXPERIMENTAL SECTION
General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. House-deionized water was purified using a Millipore Milli-Q-filtering system containing one carbon and two ion-exchange stages. All 1 H NMR spectra were recorded on a 360 MHz instrument; chemical shifts are reported in ppm relative to residual solvent. All UV spectra were recorded using a Carey 300 Bio UV-Visible spectrophotometer operating at ambient temperature. MALDI spectra were obtained using an Applied Biosystems DE-STR MALDI-TOF instrument operating in the linear, negative mode with an accuracy of (0.2%; reflector mode analyses afforded an accuracy of (0.02%) A standard buffer that was used in this work (referred to as “buffer”) was composed of 10 mM NaCl, 1.0 mM EDTA, and 2 mM PIPES, pH 7.2. The synthetic route that was used to prepare 1 is outlined in Scheme 3. 2-(Methoxycarbonyldithio)benzyl Alcohol. A solution of 1.4 g (9.99 mmol) of 2-mercaptobenzyl alcohol in 20 mL of dichloromethane was added, dropwise, to a
10.1021/bc034074m CCC: $25.00 © 2003 American Chemical Society Published on Web 11/01/2003
1192 Bioconjugate Chem., Vol. 14, No. 6, 2003
Jing et al.
Scheme 3
cooled (0 °C) solution prepared from 1 mL (10.98 mmol) of methoxycarbonylsulfenyl chloride and 60 mL of dichloromethane, under a nitrogen atmosphere. After stirring for 2 h, the mixture was washed, sequentially, with saturated sodium bicarbonate, and saturated sodium chloride, and the organic layer then dried over anhydrous MgSO4. Removal of solvent under reduced pressure, followed by column chromatographic purification [silica, chloroform/ethyl acetate (10/1, v/v)] afforded 1.20 g (52%) of 2-(methoxycarbonyldithio)benzyl alcohol having an Rf ) 0.47 and 1H NMR (CDCl3, 360 MHz) δ ppm: 7.657.27 (m, 4 H), 4.85 (s, 2 H, CH2OH), 3.83 (s, 3 H, OCH3), 2.99 (s, 1H, OH). FAB for C9H10O3S2 (M+) calcd: 230. found 230. 3,3′-Dithiopropionic Acid Bis-3-hydroxyl-1,2,3benzotriazin-4(3H)-onyl Ester. A solution was prepared from 0.500 g (2.378 mmol) of dithiopropropionic acid, 1.008 g (6.18 mmol) of 3-hydroxyl-1,2,3-benzotriazin4(3H)-one, 1.135 g (5.5 mmol) of N,N′-dicyclohexylcarbodiimide (DCC), and 20 mL of CH2Cl2. After stirring the mixture overnight at room temperature, the resulting insoluble urea was removed by filtration. The filtrate was then poured into 100 mL of a saturated aqueous NaHCO3 solution. The organic layer was separated and concentrated under reduced pressure, and the residue was purified by recrystallization from ethanol to give 1.31 g (75%) of the desired diester having 1H NMR(CD3OD): δ ppm: 8.43 (dd, 2 H), 8.41 (dd, 2 H), 8.22 (td, 2 H), 8.07 (td, 2 H), 3.40 (t, 4 H), 3.27 (t, 4 H). FAB for C20H16N6O6S2 (MH+) calcd: 501. found 501. N2,N2′-Bis[N1,N3-spermidinebischoleamideyl]-3,3′dithiopropionebisamide, Persulfate (2). To a solution
of 1.186 g (1.28 mmol) of N1,N3-spermidinebis(choleamide) in 10 mL of DMF was added 669 µL (3.84 mmol) of diisopropylethylamine (DIPEA) (2). After stirring for 10 min, 315 mg (0.63 mmol) of 3,3′-dithiopropionic acid bis3-hydroxyl-1,2,3-benzotriazin-4(3H)-one ester was directly added as a powder. After stirring overnight, the mixture was concentrated under reduced pressure. Purification by column chromatography [silica, chloroform/ methanol/water (30/9/1, v/v/v)] afforded 970 mg (75%) of the corresponding bis-sterol dimer having Rf ) 0.57 and 1H NMR (CD OD) δ ppm: 3.89 (s, 4 H), 3.74 (s, 4 H), 3 3.26 (m, 4 H), 3.14-3.11 (m, 8 H), 2.90 (brs, 4 H), 2.74 (brs, 4 H), 2.30-2.10 (m, 8 H), 2.10-2.00 (m, 8H), 1.951.00 (m, 100 H), 1.04 (s, 12 H), 0.97 (s, 12 H), 0.66 (s, 12 H). Sulfation was then carried out using standard procedures (5). Thus, 320 mg (0.148 mmol) of the bissterol dimer was dissolved in 10 mL of DMF at 0 °C, followed by direct addition of 848 mg (5.33 mmol) of Pyr‚SO3. After the mixture was stirred for 5.5 h at room temperature, 10 mL of cold water was added, followed by addition of saturated sodium bicarbonate until the pH of the mixture was 10. The combined solvent was removed under reduced pressure, and the residue was purified by column chromatography [silica, chloroform/ methanol/water (5/4/1, v/v/v)] to give 323 mg (67%) of the hexasulfated dimer (Na+ salt) having Rf ) 0.29 and 1 H NMR (CD3OD) δ ppm: 4.67 (s, 4 H), 4.45 (s, 4 H), 4.13 (s, 4 H), 3.35-3.10 (m, 16 H), 2.99 (br, 4 H), 2.83 (br, 4 H), 2.40-0.90 (m, 120 H), 0.76 (s, 12 H). MALDI for C116H184N6O54S14Na11 (M-Na) calcd: 3226. found 3231.
Molecular Umbrellas as Peptide Delivery Agents
N1,N3-(Spermidine-bis-choleamideyl)-N2-(3-dithio2-hydroxymethyl-1-phenyl)propionamide, Persulfate 3. The sulfated dimer conjugate 2 (1.81 g, 0.556 mmol) was dissolved in 20 mL of a 1.0 M phosphate buffer (pH 7.3) and mixed with 10 mL of a 1.0 M phosphate buffer containing 240 mg (0.835 mmol) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), which was adjusted to pH 7.3. After stirring for 50 min at room temperature, the solution was concentrated under reduced pressure, and the residue dissolved in 100 mL of methanol (the inorganic salts were removed by filtration). The solution, which contained the corresponding thiol monomer, was concentrated to ca. 50 mL, and 140 µL (1.0 mmol) of triethylamine then added to it. To the resulting solution was added, dropwise, a second solution, which was prepared from 307 mg (1.335 mmol) of 2-(methoxycarbonyldithio)benzyl alcohol plus 50 mL of methanol. After addition was complete, the mixture was stirred at room temperature for 5 h. Subsequent removal of solvent under reduced pressure and purification by column chromatography [silica, chloroform/methanol/ water (60/40/10, v/v/v)] afforded 804 mg (41%) of 3 having Rf ) 0.42 and 1H NMR (CD3OD) δ ppm: 7.76 (m, 1 H), 7.47 (m, 1 H), 7.30 (m, 2 H), 4.77 (s, 2 H), 4.65 (s, 2 H), 4.43 (s, 2 H), 4.12 (br, 2 H), 3.31 (m, 2 H), 3.15 (m, 6 H), 2.95 (m, 2 H), 2.73 (m, 2 H), 2.35-0.85 (m, 66 H), 0.75 (s, 6 H). MALDI (reflector mode (0.02%) for C65H99N3O28S8Na5(M-Na) calcd: 1740. found 1740.
{3-(N 2 -Propionamideyl-N 1 ,N 3 -spermidine-bischoleamideyl)}-2-dithiobenzyl-DADLE-carbamate, Persulfate 1. To a solution made from 35.3 mg (0.02 mmol) of 3 and 1 mL of DMF were added 51.2 mg (0.2 mmol) of N,N′-disuccinimidyl carbonate (DSC) and 28 µL (0.2 mmol) of triethylamine. The mixture was stirred for 15 h under a nitrogen atmosphere at room temperature, followed by removal of most of the solvent under reduced pressure; a small amount of DMF remained in order to keep the product in solution. Addition of 10 mL of acetone resulted in the precipitation of the DSC-activated ester, which was washed thoroughly with acetone, collected by filtration and freeze-dried for 2 h. This activated ester was then dissolved in 1 mL of DMF, and the resulting solution then added, dropwise, to a solution made from the sodium salt of DADLE [prepared by treating 13 mg (0.0228 mmol) of DADLE with 2 equiv of NaHCO3 in a minimum volume of water, followed by freeze-drying] plus 0.5 mL of DMF. The resulting mixture was stirred for 10 h at room temperature. The solvent was then removed under reduced pressure, and the umbrellapeptide conjugate was purified once by preparative thinlayer chromatography [silica, chloroform/methanol/water (60/40/10, v/v/v); Rf ) 0.3], twice by reverse phase preparative thin-layer chromatography [C-18 silica (VWR Scientific), methanol/water (3/2, v/v)], and one final
Bioconjugate Chem., Vol. 14, No. 6, 2003 1193
preparative thin-layer chromatography [silica, chloroform/ methanol/water (60/40/10, v/v/v)] to give 10 mg of 1 having Rf ) 0.51, and 1H NMR (CD3OD, 360 MHz) δ ppm: 7.75 (m, 1 H), 7.35-7.10 (m, 8 H), 7.08 (d, 2 H), 6.71 (d, 2 H), 5.20 (m, 2 H), 4.65 (s, 3 H), 4.45 (s, 2 H), 4.33 (m, 2 H), 4.15 (br, 3 H), 3.95 (m 2 H), 3.30 (m, 2 H), 3.15-2.85 (m, 12 H), 2.68 (m, 2 H), 2.45-0.90 (m, 72 H), 0.73 (m, 12 H). MALDI (reflector mode (0.02%) for C95H135N8O36S8Na6(M-Na) calcd: 2358. found 2358. Efflux of DADLE from Cholesterol-Rich Liposomes. A unilamellar liposomal dispersion (ca. 200 nm diameter) was prepared by standard extrusion methods using 42 mg of a mixture of POPC/POPG/cholesterol (72/ 4/24, mol/mol/mol) and 1.40 mL of buffer that was 1.0 mM in DADLE (6). The dispersion was dialyzed (three times) against 1 L of buffer for 24 h, using a dialysis tubing having a 300 kDa MW cutoff. The dispersion was then placed into a 2 mL dialysis cell, equipped with a 100 nm Nuclepore membrane. An analysis for the release of DADLE from the liposomes was made by monitoring the receiving side (23 °C) by withdrawing 0.5 mL and reacting it with 0.1 mL of a 2.0 mM fluorescamine solution in acetone for 5 min. The fluorescence intensity at 480 nm was then recorded and compared with a calibration curve. Immediately after withdrawing the 0.5 mL aliquot, 0.5 mL of fresh buffer was added to the receiving side in order to maintain a constant volume of 1.0 mL. To test for binding of DADLE to the liposomes, a related experiment was carried out in which empty liposomes were first incubated with an external solution of DADLE for 24 h, and then the resulting dispersion was dialyzed against an equal volume of buffer. In principle, if the binding of DADLE to the liposomes were negligible, then exactly 50% of the total quantity of DADLE that was added to the dispersion should be present in the receiving side of the dialysis cell, that is, the side that does not contain liposomes. Thus, 430 µL of a liposomal dispersion that was devoid of DADLE was incubated with 430 µL of a 50 µM solution of DADLE in buffer for a period of 24 h. Subsequent dialysis against 860 µL of buffer for 24 h, and analysis of the receiving side showed the presence of 50% of the total DADLE that was incubated with the liposomes. These results indicate, therefore, that there is negligible binding of DADLE to the liposomes. Reaction of 1 with GSH in Solution. A solution of 1 in buffer (1.00 mM, 0.80 mL) was mixed with 0.80 mL of 3.0 mM in glutathione in buffer at room temperature. The course of the reaction was monitored by UV (264 nm) and also by thin-layer chromatography. For UV analysis, 140 µL-aliquots were withdrawn and diluted with 240 µL of buffer, and the UV absorption was recorded. Analysis by thin-layer chromatography (silica gel, CHCl3/ CH3OH/H2O, 60/40/10, v/v/v) showed the complete disappearance of 1 (Rf 0.3) after 8 h. At that time, a portion of the liberated DADLE was isolated by thin-layer chromatography (silica gel, CHCl3/CH3OH/H2O, 65/30/2, v/v/ v) using 10 µL aliquots of the solution. The band corresponding to the free peptide (Rf 0.65) was extracted with 2 × 0.5 mL of buffer and filtered using glass wool. A 0.5 mL portion was then analyzed after reaction with 100 µL of 2 mM fluorescamine in acetone for 5 min. The fluorescence intensity at 480 nm was then compared with a calibration curve. Delivery of DADLE to the Aqueous Interior of Cholesterol-Rich Liposomes. A unilamellar liposomal dispersion (200 nm diameter) was prepared by standard extrusion methods using 60 mg of a mixture of POPC/
1194 Bioconjugate Chem., Vol. 14, No. 6, 2003 Scheme 4
POPG/cholesterol (72/4/24, mol/mol/mol) plus 2.0 mL of buffer containing 3.0 mM glutathione (6). The dispersion was dialyzed (three times) against 1 L of buffer for 28 h using dialysis tubing with a 300 kDa MW cutoff. The absence of external glutathione was confirmed by taking an 800-µL aliquot and subjecting it to dialysis for 35 min against 800 µL of buffer, using a dialysis cell that was equipped with a 100 nm Nuclepore membrane. Under these conditions, the half-life for permeation of glutathione across this membrane is 33 min. Analysis of the receiving side, by use of Ellman’s assay [5,5′dithiobis-(2nitrobenzoic acid), DTNB], indicated that
