Selective Removal of Palmitic Acid from Langmuir Monolayers by

Department of Chemistry and Biochemistry, Queens College of the City University of New. York, Flushing, New York 11367, and Department of Biochemistry...
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Langmuir 2001, 17, 5319-5323

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Selective Removal of Palmitic Acid from Langmuir Monolayers by Complexation with New Quaternary Ammonium β-Cyclodextrin Derivatives Ning Zhong,† Henna Ohvo-Rekila¨,‡ Bodil Ramstedt,‡ J. Peter Slotte,‡ and Robert Bittman*,† Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, New York 11367, and Department of Biochemistry and Pharmacy, Åbo Akademi University, P.O. Box 66, 20521 Turku, Finland Received April 17, 2001. In Final Form: May 30, 2001 The in vivo toxicity of several cyclodextrins (CyDs) appears to involve binding to and extraction of membrane cholesterol, resulting in altered membrane permeability properties. To investigate whether selective binding to lipids other than cholesterol can be achieved, a series of new water-soluble β-CyD derivatives was synthesized and tested for the ability to extract a fatty acid versus cholesterol in a model system. Quaternary ammonium derivatives (β-CyD-N+(Me)2(CH2)nOH OH- (n ) 2, 3: compounds 1-2) and β-CyD-N+(Me)2(CH2)nNMe2 OH- (n ) 1-3: compounds 3-5)) were 400-600 times more effective than unmodified β-CyD in inducing desorption of palmitic acid from Langmuir monolayers but were completely ineffective hosts for cholesterol. The rates of desorption of palmitic acid induced by amino- and hydrazineβ-CyD derivatives [β-CyD-NH(CH2)6NH2 (compound 6) and β-CyD-NHNH2 (compound 7)] were ∼100 and 10 times faster, respectively, than that induced by unmodified β-CyD. A β-CyD dimer, bis(2,2′-S-β-CyD)di(2-mercaptoethyl) ether (8), and a thioglycerol derivative of β-CyD, 6-S-(2,3-dihydroxypropyl)thio-β-CyD (9), were moderately more effective than unmodified β-CyD in inducing both palmitic acid and cholesterol desorption from their respective pure lipid Langmuir monolayers.

Introduction R-, β-, and γ-Cyclodextrins (CyDs) are cyclic oligosaccharides having six, seven, and eight R-D-glucosyl residues, respectively. Among their wide range of applications, CyDs have been used as host molecules to form inclusion complexes with hydrophobic guest molecules such as lipids1 and to enhance the bioavailability, aqueous solubility, and stability of lipophilic drugs.2-5 A serious drawback in the use of CyDs in pharmaceutical formulations is toxicity. The hemolytic activity of CyDs (and β-CyD in particular) limits their parenteral use.6,7 In some in vivo studies involving parenteral administration of β-CyD, it was concluded that renal cell damage may be a consequence of the selective extraction of cholesterol from kidney tubular membranes by CyD.8 Two prominent factors govern the ability of CyDs to serve as a lipid acceptor in an aqueous medium. First, the lipid-complexing capacity depends on the cavity size of the CyD and the structure of the lipid. The cavity size of β-CyD (7.5 Å wide, 8 Å deep) is well suited to accommodate many guest molecules. Second, the water solubility of the resulting guest-host complex depends on the solubility * To whom correspondence should be addressed. Phone: (718) 997-3279. Fax: (718) 997-3349. E-mail: [email protected]. Note: N. Z. and H. O.-R. contributed equally to this work. † City University of New York. ‡ A ° bo Akademi University. (1) Szejtli, J. Chem. Rev. 1998, 98, 1743-1753. (2) Loftsson, T.; Brewster, M. E. J. Pharm. Sci. 1996, 85, 10171025. (3) Rajewski, R. A.; Stella, V. J. J. Pharm. Sci. 1996, 85, 1142-1169. (4) Stella, V. J.; Rajewski, R. A. Pharm. Res. 1997, 14, 556-567. (5) Irie, T.; Uekama, K. J. Pharm. Sci. 1997, 86, 147-162. (6) Irie, T.; Otagiri, M.; Sunada, M.; Uekama, K.; Ohtani, Y.; Yamada, Y.; Sugiyama, Y. J. Pharmacobio-Dyn. 1982, 5, 741-744. (7) Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Pitha, J. Eur. J. Biochem. 1989, 186, 17-22. (8) Rajewski, R. A.; Traiger, G.; Bresnahan, J.; Jaberaboansari, P.; Stella, V. J.; Thompson, D. O. J. Pharm. Sci. 1995, 84, 927-932.

of the host CyD. The poor aqueous solubility of β-CyD (1.85 g per 100 mL of H2O at 25 °C)1 makes β-CyD an ineffective shuttle for the transfer of lipids from biological targets to acceptors such as lipoproteins. Therefore, β-CyD has been chemically modified to produce derivatives with very high aqueous solubility. 2,6-Di-O-methyl-β-CyD (DMβ-CyD) and 2-hydroxypropyl-β-CyD (HP-β-CyD) promote more efficient cholesterol efflux from both cells9,10 and monolayers11 than unmodified β-CyD. They have been used extensively in cell culture studies instead of serum and albumin to carry fatty acids and cholesterol (essential growth components).12-15 Although DM- and HP-β-CyD represent potential useful pharmacological agents for many purposes, including stimulation of reverse cholesterol transport in humans,16,17 their ability to efficiently extract cholesterol is likely to have detrimental effects on cell viability and cell metabolism. In fact, membrane perturbation of lysosomes on incubation of HepG2 cells with DM-β-CyD was reported recently.18 Furthermore, reduced membrane permeability function was observed (9) Kilsdonk, E. P. C.; Yancey, P. G.; Stoudt, G. W.; Bangerter, F. W.; Johnson, W. J.; Phillips, M. C.; Rothblat, G. H. J. Biol. Chem. 1995, 270, 17250-17256. (10) Neufeld, E. B.; Cooney, A. M.; Pitha, J.; Dawidowicz, E. A.; Dwyer, N. K.; Pentchev, P. G.; Blanchette-Mackie, E. J. J. Biol. Chem. 1996, 271, 21604-21613. (11) Ohvo-Rekila¨, H.; Slotte, J. P. Unpublished observations. (12) Machida, Y.; Bergeron, R.; Flick, P.; Bloch, K. J. Biol. Chem. 1973, 248, 6246-6247. (13) Kato, L.; Szejtli, J.; Szente, L. Acta Microbiol. Hung. 1993, 40, 47-58. (14) Greenberg-Ofrath, N.; Terespolosky, Y.; Kahane, I.; Bar, R. Appl. Environ. Microbiol. 1993, 59, 547-551. (15) Lo´pez-Nicola´s, J. M.; Bru, R.; Sa´nchez-Ferrer, A.; Garcı´aCarmona, F. Biochem. J. 1995, 308, 151-154. (16) Atger, V. M.; de la Llera Moya, M.; Stoudt, G. W.; Rodrigueza, W. V.; Phillips, M. C.; Rothblat, G. H. J. Clin. Invest. 1997, 99, 773780. (17) Christian, A. E.; Byun, H.-S.; Zhong, N.; Wanunu, M.; Marti, T.; Furer, A.; Diederich, F.; Bittman, R.; Rothblat, G. H. J. Lipid Res. 1999, 40, 1475-1482.

10.1021/la010565o CCC: $20.00 © 2001 American Chemical Society Published on Web 07/12/2001

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on incubation of intestinal and nasal epithelial cells with DM- and HP-β-CyDs.19-21 The need to prepare safer CyD derivatives as pharmaceutical agents has been a goal of several previous studies.5 For example, the hemolytic activity of unmodified CyD was diminished significantly on the introduction of negatively charged groups into the hydroxyl groups of CyD, as in sulfobutyl-β-CyD and β-CyD-sulfate22,23 and by introduction of acetyl groups into DM-β-CyD.24 In the present study, we modified one of the primary-side hydroxyl groups of β-CyD to improve its water solubility by introducing a quaternary ammonium, thioglycerol, aminoalkyl, or hydrazine group, and we tested the ability of these derivatives to form a complex with cholesterol and palmitic acid in monolayers. We found that quaternary ammonium β-CyDs (compounds 1-5) promote the highly efficient desorption of palmitic acid but do not cause significant leakage of cholesterol from Langmuir monolayers. Materials and Methods Materials. Hydrazine was purchased from Fluka. Cholesterol, palmitic acid, and R-thioglycerol were purchased from Sigma. 1,6-Diaminohexane, NaCNBH3, 2-dimethylamino-1-ethanol, N,N,N′,N′-tetramethyldiaminoethane, and 3-dimethylamino-1propanol were obtained from Aldrich. N,N,N′,N′-Tetramethyldiaminomethane was from Matheson Coleman & Bell. N,N,N′,N′Tetramethyl-1,3-propanediamine was from Acros. Bis(2-mercaptoethyl) ether and β-CyD were obtained from TCI America. Dess-Martin periodinane,25 β-CyD-6-deoxy-aldehyde,26 and β-CyD-6-deoxytosylate (Ts-β-CyD)27,28 were made according to previously reported methods. TLC was carried out on silica gel plates (E. Merck 60-F254) in 1-propanol/water/EtOAc/concentrated NH4OH ) 5:3:1:1. HPLC was carried out in a BioCad Sprint perfusion chromatography system using an amino column (Alltech, 4.6 × 250 mm), a flow rate of 1.0 mL/min, a mobile phase of 80% CH3CN/H2O, and UV detection at 190 nm. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. FAB MS and MALDI MS were run in a glycerol matrix at Michigan State University; electrospray MS was run at Hunter College of CUNY. Removal of Monolayer Lipids to the Subphase by β-CyD Derivatives. Monolayers containing pure cholesterol or pure palmitic acid were prepared at the air/water interface at 22 °C and compressed to a lateral surface pressure of 20 mN/m. The trough used was a zero-order type, with a reaction chamber (28 mL volume, 28.3 cm2 area) separated by a glass bridge from the lipid reservoir. After a stable baseline had been obtained, β-CyD or its analogue was injected into the stirred reaction chamber without penetrating the monolayer (in a volume not exceeding 1 mL). The final concentration of the β-CyD derivatives in the reaction chamber was varied, as indicated. The removal of monolayer lipids to the subphase was determined from the area decrease of monolayer at constant surface pressure as described (18) Jadot, M.; Andrianaivo, F.; Dubois, F.; Wattiaux, R. Eur. J. Biochem. 2001, 268, 1392-1399. (19) Shao, Z.; Li, Y.; Chermak, T.; Mitra, A. K. Pharm. Res. 1994, 11, 1174-1179. (20) Matsubara, K.; Abem, K.; Irie, T.; Uekama, K. J. Pharm. Sci. 1995, 84, 1295-1300. (21) Totterman, A. M.; Schipper, N. G.; Thompson, D. O.; Mannermaa, J. P. J. Pharm. Pharmacol. 1997, 49, 43-48. (22) Macarak, E. J.; Kumor, K.; Weisz, P. B. Biochem. Pharmacol. 1991, 42, 1502-1503. (23) Shiotani, K.; Uehata, K.; Irie, T.; Uekama, K.; Thompson, D. O.; Stella, V. J. Pharm. Res. 1995, 12, 78-84. (24) Hirayama, F.; Mieda, S.; Miyamoto, Y.; Arima, H.; Uekama, K. J. Pharm. Sci. 1999, 88, 970-975. (25) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899. (26) Cornwell, M. J.; Huff, J. B.; Bieniarz, C. Tetrahedron Lett. 1995, 36, 8371-8374. (27) Zhong, N.; Byun, H.-S.; Bittman, R. Tetrahedron Lett. 1998, 39, 2919-2920. (28) Byun, H.-S.; Zhong, N.; Bittman, R. Org. Synth. 1999, 77, 225230.

Zhong et al. Scheme 1. Syntheses of Compounds 1-5

previously.29 From the mean molecular area at a given surface pressure and temperature, the concentration of cholesterol or palmitic acid removed was calculated as a function of time. Aqueous Solubility of β-CyD Derivatives. A suspension of CyDs (0.1 g) in 1 mL of H2O was sonicated in a model FS28H sonicator (Fisher Scientific) for 30 min and then stirred in a thermostated bath (25 °C) overnight. When complete dissolution of the CyD had not occurred, the mixture was filtered through a 0.45-µm Teflon Cameo filter, and the filtrate was lyophilized. The mass was measured as a function of the volume. Syntheses of β-CyD-N+(Me)2(CH2)nOH OH- (n ) 2 and 3: Compounds 1 and 2, Scheme 1). Ts-β-CyD (1.0 g, 0.77 mmol) and either 2-dimethylamino-1-ethanol (7.8 mL, 77 mmol) or 3-dimethylamino-1-propanol (9.6 mL, 77 mmol) was added to a reaction flask containing 100 mL of dry pyridine under N2. The reaction mixture was stirred at 60 °C for 24 h. Pyridine was removed by vacuum evaporation. The residue was dissolved in 30 mL of H2O and then applied to a CM-Sephadex C-25 column (eluted with 0-0.4 M NH4HCO3). The fraction containing the product was collected and concentrated. Small molecules were removed on a Sephadex G-15 column (elution with H2O). Removal of the water gave the crude product, which was dissolved in a minimum volume of H2O and applied to a mixed-bed ion-exchange (Amberlite MB3) column (elution with H2O). Lyophilization provided the pure product as a white powder; TLC Rf ) 0.18 (for both 1 and 2). Compound 1 (n ) 2): 0.94 g (94% yield); 1H NMR (D2O) δ 3.07 (s, 3H), 3.09 (s, 3H), 3.35-3.94 (m, ∼46H), 4.894.97 (m, 7H); 13C NMR δ 32.9, 41.0, 50.6, 51.1, 53.6, 58.5, 58.6, 58.8, 59.1, 64.1, 64.9, 65.3, 69.3, 69.9, 70.2, 70.4, 70.5, 70.8, 71.1, 71.3, 71.4, 77.5, 79.1, 79.2, 79.4, 79.9, 81.3, 97.8, 98.6, 99.8, 100.0, 100.1, 100.3. MALDI MS: C46H81O35N, calcd for [M - OH + H]+ 1207.46, found 1207.15. Compound 2 (n ) 3): 0.91 g (90% yield); 1H NMR (D O) δ 1.42 (br s, 2H), 2.10 (br s, 2H), 2.30 (br s, 3H), 2 2.56 (br s, 3H), 2.67 (m, 2H) 2.78-3.51 (m, ∼42H), 3.88-4.03 (m, 2H), 4.41-4.60 (m, 5H); 13C NMR δ 23.5, 23.8, 33.3, 41.4, 50.3, 50.6, 50.8, 53.8, 56.7, 56.8, 57.2, 59.1, 59.4, 61.81, 67.9, 68.1, 69.6, 69.8, 70.2, 70.3, 70.5, 70.9, 71.1, 71.4, 71.6, 71.8, 72.3, 72.6, 74.2, 74.3, 79.3, 79.4, 79.6, 80.1, 94.2, 94.3, 96.3, 98.9, 100.1, 100.3, 100.6. Electrospray MS: C47H82O35N, calcd for [(M - OH)]+ 1220.5, found 1220.6. Syntheses of β-CyD-N+(Me)2(CH2)nN(Me)2 OH- (n ) 1-3: Compounds 3-5, Scheme 1). These compounds were prepared by the method used to prepare compounds 1 and 2, except that the amino-containing starting materials for compounds 3, 4, and 5 were N,N,N′,N′-tetramethyldiaminomethane (10.6 mL, 77 mmol), N,N,N′,N′-tetramethyldiaminoethane (11.7 mL, 77 mmol), and N,N,N′,N′-tetramethyl-1,3-propanediamine (10.1 g, 77 mmol), respectively. The pure product was obtained as a white powder; TLC Rf ) 0.18 (for 3, 4, and 5). Compound 3 (n ) 1): 0.95 g (94% yield); 1H NMR (D2O) δ 2.79 (s, 6H), 3.22 (s, 3H), 3.23 (s, 3H), 3.40-3.98 (m, ∼44H), 4.96-5.08 (m, 7H); 13C NMR δ 43.3, 49.4, 51.8, 52.4, 59.2, 59.7, 59.8, 59.9, 60.0, 60.6, 66.6, 70.9, 71.05, 71.09, 71.12, 71.25, 71.33, 71.38, 71.44, 71.6, 71.9, 72.0, 72.3, 72.45, 72.54, 72.7, 78.67, 80.3, 80.4, 80.6, 80.7, 82.4, 99.8, 101.0, 101.1, 101.2, 101.3, 101.5. MALDI MS: C47H85O35N2, calcd for [M + H]+ 1237.49, found 1237.17. Compound 4 (n ) 2): 0.95 g (93% yield); 1H NMR (D2O) δ 1.89 (s, 6H), 1.92 (m, 1H), 2.21 (m, 1H), 2.79 (m, 2H), 2.94 (m, 6H), 3.11-4.16 (m, ∼42H), 4.88-5.28 (m, 7H); 13C NMR δ 31.1, 36.7, 38.5, 44.6, 45.2, 45.3, 46.5, 47.2, 54.6, 59.8, 69.5, 71.5, 71.8, 72.7, 72.9, 80.5, 80.8, 82.8, 101.3, 101.7. MALDI MS: C48H84O34N2, calcd for [M - H2O]+ 1232.49, found 1232.98. Compound 5 (n ) 3): 0.97 g (94% yield); 1H NMR (D2O) δ 1.76 (br s, 2H), 1.85 (s, 6H), 2.42-2.52 (m, 6H), 2.66 (br (29) Ohvo, H.; Slotte, J. P. Biochemistry 1996, 35, 8018-8024.

Selective Removal of Palmitic Acid from Monolayers Scheme 2. Synthesis of 6

Scheme 3. Synthesis of 7

Scheme 4. Synthesis of 8

s, 2H), 2.78 (m, 2H), 3.30-4.15 (m, ∼42H), 4.85-5.12 (m, 7H); 13C NMR δ 23.3, 44.2, 60.3, 61.3, 61.9, 63.1, 71.6, 72.3, 72.8, 72.9, 73.1, 73.4, 73.5, 73.8, 74.0, 74.1, 81.9, 82.1, 82.8, 84.1, 102.5, 102.9, 103.0, 103.8. FAB MS: C49H88O35N2, calcd for [M]+ 1264.52, found 1264.64. β-CyD-NH(CH2)6NH2 (Compound 6, Scheme 2). To a solution of Ts-β-CyD27,28 (2.31 g, 2.0 mmol) and 1,6-diaminohexane (9.76 g, 83 mmol) in 20 mL of DMF was added K2CO3 (1.6 g, 11 mmol). After the reaction mixture was stirred at 55 °C for 2 days, the crude product was collected by pouring the mixture into 500 mL of ethanol. Small molecules were removed on a G-15 Sephadex column using H2O as the eluent. The included 1,6-diaminohexane was removed by heating the product in ethanol overnight at reflux. Evaporation of the solvents gave the pure product as a white powder: 2.2 g (89% yield); TLC Rf ) 0.40; 1H NMR (D2O) δ 2.47 (br s, 2H), 2.61-2.77 (m, 4H), 2.86-2.98 (m, 4H), 3.11 (m, 1H), 3.31 (m, 1H), 3.44-3.76 (m, ∼42H), 4.86-5.00 (m, 7H); 13C NMR δ 39.7, 40.0, 41.3, 60.1, 72.0, 73.2, 81.0, 101.9, 119.9. Electrospray MS: C48H85O34N2, calcd for [M + H]+ 1233.5, found 1233.2. FAB MS: C48H85O34N2, calcd for [M + H]+ 1233.50, found 1233.38. Synthesis of β-CyD-NHNH2 (Compound 7, Scheme 3). β-CyD (1.85 g, 1.63 mmol) was dissolved in 45 mL of DMF. After the Dess-Martin periodinane25 (DMP, 1.0 g, 2.4 mmol) was added, the reaction mixture was stirred for 2 h at room temperature. Addition of 1.5 L of acetone at -10 °C afforded crude β-CyD monoaldehyde by filtration.25 The monoaldehyde was dissolved in 50 mL of H2O, H2NNH2 (0.24 mL, 8.0 mmol) was added, and the pH was adjusted to 6.0 with 1 M HCl. After 6 h, NaCNBH3 (0.11 g, 1.8 mmol) was added. The reaction mixture was stirred for 1 week. The crude product was purified by chromatography on a Sephadex G-15 column (eluted with 150 mL of H2O), then on a CM-Sephadex C-25 column (gradient elution with 0-0.02 M NH4OH, 500 mL), and then by HPLC, yielding 0.35 g (19% overall yield from β-CyD): TLC Rf ) 0.15; HPLC tR ) 4.16 min (tR of β-CyD was 4.80 min); 1H NMR (D2O) δ 3.47-4.16 (m, ∼42H), 4.95 (br s, 7H), 5.28 (br s, 2H); 13C NMR δ 43.3, 60.6, 61.8, 62.0, 63.3, 71.9, 72.2, 73.4, 73.5, 73.8, 74.1, 74.3, 74.4, 82.0, 82.1, 83.2, 84.3, 102.9, 103.1, 103.3, 103.6. Electrospray MS: C42H79O38N2, calcd for [M + 4H2O - H]+ 1219.4, found 1219.2. Bis(2,2′-S-β-CyD)-bis(2-mercaptoethyl) Ether (Compound 8, Scheme 4). To a solution of Ts-β-CyD (2.5 g, 1.9 mmol) in 30 mL of DMF was added K2CO3 (0.85 g, 6.0 mmol). After the solution was stirred at room temperature, bis(2-mercaptoethyl) ether (0.14 g, 0.90 mmol) was added, and the temperature was

Langmuir, Vol. 17, No. 17, 2001 5321 Scheme 5. Synthesis of 9

increased to 60 °C. After 2 days, the reaction mixture was poured into 500 mL of acetone, giving a precipitate that was dissolved in 50 mL of H2O. The product was purified by column chromatography on Sephadex LH-20 [elution with 2-PrOH/H2O (1:1)], giving 0.82 g (18%) of the product as a white powder: TLC Rf ) 0.2; 1H NMR (D2O) δ 2.49-2.63 (m, 4H), 2.78-2.81 (m, 2H), 3.25-3.31 (m, 2H), 3.48-4.03 (m, ∼84H), 4.97-5.04 (m, 14H); 13C NMR δ 21.7, 58.2, 62.2, 68.6, 69.5, 69.8, 70.0, 70.2, 71.0, 71.5, 78.8, 79.2, 79.4, 99.5, 99.9, 100.1. MALDI MS: C88H145O69S2, calcd for [M - H]+ 2369.73, found 2369.41; C88H146O69S2Na, calcd for [M + Na]+ 2393.72, found 2394.12; C88H146O69S2K, calcd for [M + K]+ 2409.70, found 2410.15. 6-S-(2,3-Dihydroxypropyl)-6-thio-β-CyD (Compound 9, Scheme 5). To a solution of Ts-β-CyD (1.24 g, 0.90 mmol) in 5 mL (58 mmol) of R-thioglycerol was added K2CO3 (1.4 g, 10 mmol). The reaction mixture was stirred at 60 °C until the starting material was consumed (about 1 day). The reaction mixture was poured into 400 mL of ethanol, and the resulting residue was collected by filtration. The residue was washed with ethanol, giving the product (1.0 g, 0.82 mmol) as a white powder: yield, 91%; TLC Rf ) 0.57; 1H NMR (D2O) δ 2.60 (m, 1H), 2.70-2.75 (m, 2H), 3.08 (d, 1H, J ) 13.4 Hz), 3.47-3.90 (m, ∼43H), 4.975.20 (m, 7H);13C NMR δ 35.3, 37.0, 37.2, 61.8, 62.1, 65.9, 72.2, 72.5, 72.7, 73.3, 73.5, 73.6, 74.5, 74.6, 82.7, 82.7, 85.6, 85.7, 103.0, 104.4. FAB MS: C45H77O36S, calcd for [M + H]+ 1225.38, found 1225.13.

Results and Discussion Synthesis. Quaternary ammonium β-CyD derivatives 1-5 were prepared in ∼94% yield from Ts-β-CyD27,28 via SN2 displacement using tertiary amines in pyridine and were isolated easily by using a series of chromatographic steps (Scheme 1). Amino β-CyD derivative 6 was synthesized in a similar fashion in DMF/K2CO3 in 89% yield (Scheme 2). The included 1,6-diaminohexane was removed from the product by heating the complex in ethanol overnight. Scheme 3 shows the synthesis of β-CyD hydrazine derivative 7; β-CyD monoaldehyde, prepared by Dess-Martin oxidation of β-CyD,25 was reacted with hydrazine to give the corresponding Schiff base. The latter was reduced with NaCNBH3 in situ. After the byproducts were removed on a Sephadex G-15 column, unreacted β-CyD was removed on a CM-Sephadex C-25 column using gradient eluent (0-0.02 M NH4OH). The final purification was carried out on HPLC, giving 7 in 19% overall yield. Scheme 4 shows the synthesis of β-CyD thio derivative dimer 8, which was obtained in 18% yield. DMF was removed by precipitation in acetone. The by-products (βCyD and probably the monomer) were removed on a Sephadex LH-20 column, eluting with 2-PrOH/H2O (1:1). β-CyD thio derivative 9 was prepared in 91% yield using R-thioglycerol as both the thiol reagent and the solvent (Scheme 5). Ts-β-CyD was completely converted to the product as shown by TLC. Excess R-thioglycerol was removed by precipitation in ethanol. Desorption of Cholesterol from Monolayers. Table 1 shows the rates of cholesterol desorption from monolayer membranes on injection of the synthetic β-CyD derivatives into the subphase. Because of the limited quantities of β-CyDs available for these studies, the β-CyD concentration in the reaction chamber was varied. The highest possible concentration was used for the β-CyD derivatives that were poor promoters of cholesterol efflux. At some point, the solubility of the β-CyD became limiting, since β-CyD was injected from a concentrated solution (the

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Table 1. Rates of Desorption of Cholesterol from Monolayersa compound

concn (mM)

desorption rate [pmol/(cm2 min)]

rate relative to β-CyD

β-CyD β-CyD 1 2 3 4 5 6 7 8 9 β-CyD-S-β-CyD

0.2 0.8 1.3 1.0 1.0 1.3 1.3 0.8 0.2 0.2 0.8 0.1

0.8 ( 0.04 8.9 ( 0.2 0 0 0 0 0 2.8 ( 0.1 8.3 ( 0.6 11.3 ( 0.1 22.5 ( 0.5 25.5 ( 1.2

1 1

0.3 10.5 14.3 2.5 85b

a

The desorption of cholesterol was described in the experimental procedures. Values are means ( standard error of the mean from two or three experiments. b The desorption rate of cholesterol is not a simple linear function of the subphase β-CyD concentration (ref 29). Figure 2 in ref 29 was used and extrapolated to give a desorption rate at 0.1 mM β-CyD of approximately 0.3 pmol/(cm2 min). Using this value, the desorption rate of β-CyD-S-β-CyD relative to β-CyD was calculated to be 85.

maximum volume of β-CyD injected into the 28-mL reaction chamber was 1 mL). To compare the rates relative to that mediated by unmodified β-CyD, the rates of cholesterol desorption are shown at two β-CyD concentrations (Table 1). The short and bulky side chains of our β-CyD derivatives are not expected to be accommodated in the β-CyD cavity.30 Since the depth of a dimer cavity is closely matched with the length of the long axis of the cholesterol molecule (∼15 Å), it is not surprising that complexes with stoichiometries of 1 steroid per 2 β-CyDs31,32 have been noted, as well as other stoichiometries. β-CyD-S-β-CyD, a β-CyD dimer, was found to bind cholesterol more strongly than the corresponding monomer.33 Table 1 shows that β-CyDS-β-CyD was more efficient in inducing cholesterol desorption than any of the other derivatives used in the present study. Of the new β-CyD derivatives described in this report, bis(2,2′-S-β-CyD)-di(2-mercaptoethyl) ether (8) and β-CyD-NHNH2 (7) were the most efficient cholesterol acceptors, inducing cholesterol desorption 10-14 times faster than unmodified β-CyD. The thio-β-CyD monomer, 6-S-(2,3-dihydroxypropyl)-6-thio-β-CyD (9), induced cholesterol desorption from monolayers with a rate 6-fold lower than that of dimer 8. Of the amino-β-CyD derivatives used in this study, β-CyD-NHNH2 (7) was the most efficient in promoting cholesterol efflux, as noted above. When a series of methylene groups was inserted between the two nitrogens in the side chain, the cholesterol-complexing ability diminished. β-CyD-N+(Me)2(CH2)nOH OH- (n ) 2 and 3, compounds 1 and 2) did not induce desorption of cholesterol from monolayers, perhaps because the quaternary ammonium linkage interferes sterically with the inclusion of cholesterol in the β-CyD cavity. The other synthetic quaternary ammonium derivatives, β-CyD-N+(Me)2(CH2)nN(Me)2 OH- (n ) 1-3, compounds 3-5) also did not promote efflux of cholesterol from monolayers. In a related study, short-chain β-CyD-amines (β-CyD-N+H3, (30) Liu, Y.; Han, B.-H.; Li, B.; Zhang, Y.-M.; Zhao, P.; Chen, Y.-T.; Wada, T.; Inoue, Y. J. Org. Chem. 1998, 63, 1444-1454. (31) Cabrer, P. R.; Alvarez-Parrilla, E.; Meijide, F.; Seijas, J. A.; Nu´n˜ez, E. R.; Tato, J. V. Langmuir 1999, 15, 5489-5495. (32) Cserha´ti, T.; Forga´s, E. J. Pharm. Biomed. Anal. 2000, 22, 2531. (33) Breslow, R.; Zhang, B. J. Am. Chem. Soc. 1996, 118, 84958496.

Table 2. Rates of Desorption of Palmitic Acid from Monolayersa compound

concn (mM)

desorption rate [pmol/(cm2 min]

rate relative to β-CyDb

β-CyD 1 2 3 4 5 6 7 8 9 β-CyD-S-β-CyD

0.1 0.01 0.01 0.01 0.01 0.01 0.01 0.1 0.01 0.1 0.05

7.0 ( 0.1 310.7 ( 8.2 448.2 ( 3.5 316.7 ( 5.2 310.8 ( 10.5 309.4 ( 11.5 78.2 ( 5.1 150.3 ( 2.5 4.1 ( 0.1 48.9 ( 0.0 92.5 ( 3.5

1 443.9 640.3 452.5 443.9 442.0 111.7 21.5 5.8 7.0 26.4

a Pure palmitic acid monolayers were spread at the air/water interface. The monolayer was compressed at 22 °C and held at a constant lateral surface pressure (20 mN/m). The rate of desorption to the subphase containing β-CyD was determined from the rate of monolayer area decrease. Values are averages ( standard error of the mean from two or three experiments. b When calculating the relative rates, we made the assumption that the desorption rate dependence is linearly correlated with the β-CyD concentration, although this assumption may not be true under all conditions used.

β-CyD-N+H(Me)2, and β-CyD-N+H(Et)2) were found to be less effective than DM-β-CyD in promoting cholesterol efflux from cells.17 Desorption of Palmitic Acid from Monolayers. Palmitic acid was selected to test the ability of β-CyD derivatives because it forms stable monolayers and is also known to bind to CyDs.15,34,35 All of the β-CyD derivatives were more efficient than unmodified β-CyD in inducing palmitic acid desorption from monolayer membranes (Table 2). With many of the β-CyD derivatives we used, the desorption rate was too fast to measure at a concentration of 0.10 mM; therefore, the concentration of the β-CyD derivative was reduced to 0.01 mM. However, at a concentration of 0.01 mM unmodified β-CyD, no palmitate efflux was observed. Therefore, when calculating the relative rates, we assumed that there is a linear dependence of the desorption rate on β-CyD concentration, although this assumption may not be true under all conditions used. β-CyD-N+(Me)2(CH2)3OH OH- (compound 2) induced palmitic acid desorption most efficiently (Table 2), perhaps because of electrostatic interactions between the positive charge on the β-CyD derivative and the negative charge in deprotonated palmitic acid.36 Earlier studies showed that the included molecules are normally oriented in the host in such a position that maximum contact between the hydrophobic part of the guest and the apolar β-CyD cavity is achieved.37 If it is sterically possible, the hydrophilic part of the guest molecule remains at the outer face of the complex, as in palmitic acid. The other quaternary ammonium β-CyD derivatives (compounds 1, 3, and 5) also induced a very fast desorption of palmitic acid (∼450-fold faster than that induced by β-CyD). The amino derivative β-CyD-NH(CH2)6NH2 (compound 6) was about 100-fold more efficient in inducing palmitic acid desorption from monolayers (Table 2) than unmodified β-CyD but was ∼4-fold less efficient than the quaternary ammonium derivatives. The hydrazine derivative 7 was (34) Schlenk, H.; Sand, D. M. J. Am. Chem. Soc. 1961, 83, 23122320. (35) Slotte, J. P.; Illman, S. Langmuir 1996, 12, 5664-5668. (36) Rekharsky, M. V.; Mayhew, M. P.; Goldberg, R. N.; Ross, P. D.; Yamashoji, Y.; Inoue, Y. J. Phys. Chem. B 1997, 101, 87-100. (37) Szejtli, J. In Cyclodextrin Technology; Kluwer: Dordrecht, The Netherlands, 1988; Chapters 2 and 3.

Selective Removal of Palmitic Acid from Monolayers

Langmuir, Vol. 17, No. 17, 2001 5323

Table 3. Aqueous Solubility of β-CyDs at 25 °Ca

The ether-linked derivative bis(2,2′-S-β-CyD)-bis(2-mercaptoethyl) ether (8) had a higher water solubility than β-CyD. There seems to be a relationship between good water solubility of the β-CyD compound and its ability to induce palmitic acid desorption from monolayers (Table 2). On the other hand, the ability to induce cholesterol desorption is not correlated with the water solubility of the β-CyD derivative.

compound β-CyD 1 2 3 4 5 6 7 8 9 β-CyD-S-β-CyD

solubility mM g/100 mL 16.3 330 304 334 330 227 146 139 19.0 51.5 14.6

1.85b 43.0 40.0 44.0 43.0 30.5 18.0 16.0 4.5 6.3 3.3

solubility relative to β-CyD 1 20.2 21.6 20.5 20.2 13.9 9.7 8.6 2.4 3.4 0.9

a The β-CyD derivative (100 mg) was added to 1 mL of water, and the mixture was sonicated for 30 min and then stirred overnight at 25 °C. The mixture was filtered, and the filtrate was lyophilized. The mass was measured as a function of the volume. The results are the average of two or three experiments. b Data taken from ref 1.

5-fold less effective in inducing palmitic acid desorption than 6. The derivatives with a nitrogen-containing group induced a faster rate of palmitic acid desorption than the thio-β-CyD derivatives (Table 2). 6-S-(2,3-Dihydroxypropyl)-6-thio-β-CyD (9) and bis(2,2′-S-β-CyD)-bis(2-mercaptoethyl) ether (8) were the least efficient palmitic acid acceptors, while β-CyD-S-β-CyD was slightly better. Role of Aqueous Solubility of β-CyD Derivatives in Lipid Efflux Kinetics from Monolayers. All of the β-CyD derivatives were more water soluble than unmodified β-CyD (Table 3). As expected, the quaternary ammonium group greatly enhanced the water solubility. The other amino-β-CyD derivatives (β-CyD-NH(CH2)6NH2 and β-CyD-NHNH2) were somewhat less water soluble than the quaternary ammonium CD derivatives but were still 5-8 times more soluble than unmodified β-CyD (Table 3).

Conclusion Addition of highly water soluble quaternary ammonium β-CyD derivatives to the subphase of lipid monolayers induced highly efficient desorption of palmitic acid but no extraction of cholesterol. Instead, cholesterol desorption was most efficiently induced by β-CyD dimers. Palmitic acid, on the other hand, underwent desorption from monolayers with equal rates on adding uncharged β-CyD monomer 9 and dimer 8 to the subphase. Our results indicate that it is possible to synthesize β-CyD derivatives with different lipid-complexing capabilities; therefore, a goal of future work is to selectively extract a specific target lipid from a more complex system, such as biological membranes. Acknowledgment. This research was supported by generous grants from the Academy of Finland, the Sigrid Juselius Foundation, the Oskar O ¨ flund Foundation, the Borg Foundation, the Magnus Ehrnrooth Foundation, the Svenska Kulturfonden Foundation, the Medicinska Understo¨dfo¨reningen Liv och Ha¨lsa Foundation, the Walter and Lisi Wahl Foundation, and the A° bo Akademi University. Support for this work by NIH Grant HL-16660 is gratefully acknowledged. We thank Dr. H.-S. Byun for helpful discussions. LA010565O