Ion Conductors Derived from Biogenic Amines, Bile Acids, and Amino

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Bioconjugate Chem. 2002, 13, 1314−1318

Ion Conductors Derived from Biogenic Amines, Bile Acids, and Amino Acids Punam Bandyopadhyay, Prasun Bandyopadhyay, and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015. Received May 24, 2002; Revised Manuscript Received August 20, 2002

A family of conjugates has been synthesized from spermine, putrescine, lysine, γ-aminobutyric acid, sarcosine, cholic acid, glycocholic acid, 3R,7R-dihydroxycholic acid, and 3R,12R-dihydroxycholic acid, based on a design principle previously reported (Bandyopadhyay, P., Janout, V., Zhang, L., Regen, S. L. (2001) J. Am. Chem. Soc. 123, 7691). Each of these conjugates was found to exhibit significant activity in promoting the transport of Na+ across liposomal membranes derived from 1,2-dimyristoleoylsn-glycero-3-phosphocholine, and also from 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine. In all cases, plots of pseudo first-order rate constants, kobsd vs (mol % of ion conductor)2 were found to be linear, indicating that transport-active dimers are involved and that only a small fraction of the conjugates are in an aggregated form. An operational comparison that has been made within this series of conjugates indicates that Na+ transport activity and membrane selectivity have a moderate dependency on the composition and the structure of the ion conductor.

INTRODUCTION

We have been involved in the synthesis of new classes of ion conductors (i.e., molecules that transport ions across phospholipid bilayers) over the past several years (1). Our motivation for such work stems from belief that those ion conductors, which exhibit high activity and high membrane selectivity, have potential as antibiotics. In particular, ion conductors that are capable of selectively killing microorganisms in the presence of mammalian cells, by destroying their membrane integrity, would circumvent two of the major mechanisms of drug resistance; that is, enzymatic degradation within the cell and export pathways (2, 3). Recently, we introduced a new design principle for the construction of ion conductors in which two or more facial amphiphiles are covalently attached to a linear backbone (Scheme 1) (1k,l). When incorporated into a lipid bilayer, such conjugates are expected to favor conformation B where each hydrophobic face (darkened rectangle) is in intimate contact with the alkyl chains of neighboring phospholipids, and each hydrophilic face (lightly shaded rectangle) points toward a hydrophilic face of a nearestneighbor. Conformation A, on the other hand, is expected to be favored when the conjugate is adsorbed to the membrane surface. In principle, dimerization of B across a lipid bilayer should produce a contiguous pathway that permits a flow of ions. One prototype that was recently reported (i.e., 1) exhibited ion conducting and monolayer properties that were consistent with this model. As noted previously, 1 is unique in the sense that it is derived from the direct conjugation of two underivatized biogenic precursorssspermine and cholic acid. In the work that is reported herein, we sought to expand this class of ion conductors by preparing a series

of analogues; that is, a variety of ion conductors from naturally occurring facial amphiphiles and biogenic amines or amino acids. Our primary interest in this study was to determine the sensitivity of ion transport activity and membrane selectivity toward modest changes in the structure and composition of the ion conductor. With this aim in mind, we focused on the synthesis of a series of conjugates derived from spermine, putrescine, lysine, γ-aminobutyric acid, sarcosine, cholic acid, glycocholic acid, 3R,7R-dihydroxycholic acid, and 3R,12Rdihydroxycholic acid, and a determination of their ability to promote Na+ transport across liposomal bilayers made from 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine [(C14: 1)PC], and also from 1,2-dipalmitoleoyl-sn-glycero-3phosphocholine [(C16:1)PC]. As discussed previously, the Scheme 1

* To whom correspondence should be addressed. E-mail: [email protected].

10.1021/bc0255539 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/19/2002

Ion Conductors Derived from Biogenic Amines

ability of an ion conductor to respond to differences in membrane thickness raises the possibility of being able to exploit subtle differences between the membranes of mammals and of microorganisms from a therapeutic standpoint (1j). In particular, fungal and bacterial membranes are expected to have regions that are thinner and more susceptible toward ion transport than mammalian membranes, since the cholesterol that is present in the latter is known to condense and thicken bilayers.

MATERIALS AND METHODS

General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. Phospholipids that were used for preparing liposomes were obtained from Avanti Polar Lipids (Alabaster, AL). Specific procedures for preparing a paramagnetic shift reagent, for forming vesicles, and for measuring rates of Na+ transport by NMR spectroscopy have previously been described in detail (1l). House-deionized water was purified using a Millipore Milli-Q-filtering system containing one carbon and two ion-exchange stages. Vesicle extrusions were carried out using a Lipex Biomembrane apparatus (Vancouver, BC). All 1H NMR and 23Na+ NMR spectra were recorded on 500 and 360 MHz instruments, respectively; chemical shifts are reported in ppm relative to residual solvent. The pH of the NaCl solution that was used in these transport experiments was typically ca. 6. Conjugate 2. To a solution of glycocholic acid (0.200 g, 0.43 mmol) in 1 mL of DMF was added 0.10 mL of diisopropylethylamine (DIPEA) plus O-(3,4-dihydro-4oxo-1,2,3-benzotriazin-3-yl)-N,N,N′N′-tetramethyluroni-

Bioconjugate Chem., Vol. 13, No. 6, 2002 1315

um tetrafluoroborate (0.163 g, 0.47 mmol). After stirring this mixture for 2 h at room temperature, a solution of spermine (18.2 mg, 0.090 mmol) and DIPEA (0.3 mL) in 1 mL DMF was added, dropwise, followed by additional stirring for 16 h at 55 °C. Removal of solvent under reduced pressure at 50 °C afforded a residue, which was then dissolved in 1 mL of methanol. This solution was then added to 15 mL of a 10% aqueous NaHCO3 solution, and the solid collected via filtration and purified by column chromatography (SiO2, CHCl3/MeOH/H2O, 120/ 60/2, v/v/v) affording 80 mg (47%) of 2 having Rf 0.45 (SiO2, CHCl3/MeOH/H2O, 120/60/2, v/v/v) and 1H NMR (CD3OD, 500 MHz) δ (ppm): 4.07 (m, 4 H), 3.95 (bs, 4 H) 3.79 (m, 8 H), 3.35 (m, 4 H), 3.30-3.10 (m, 12 H), 2.21-0.91 (m, 128 H), 0.71 (s, 12 H). HRMS for C114H190N8O20 (MNa+): Calcd: 2014.3989.Found: 2014.4086. Conjugate 3. A solution was prepared from cholic acid succinimide ester (2.0 g, 4 mmol), 10 mL of DMF, 2 mL of DIPEA, and 4-aminobutyric acid (0.404 g, 4 mmol) and stirred at 40 °C for 24 h. Removal of solvent under reduced pressure afforded a residue that was dissolved in 1.5 mL of methanol and then precipitated via the addition of 15 mL of a 10% aqueous NaHCO3 solution. The resulting solid was filtered and purified via column chromatography (SiO2, CHCl3/MeOH/H2O, 120/60/1, v/v/ v) yielding 1.0 g (55%) of the corresponding 4-aminobutyric acid conjugate of cholic acid having Rf 0.50 (SiO2, CHCl3/MeOH/H2O, 120/60/1, v/v/v) and 1H NMR (CD3OD 500 MHz) δ (ppm): 3.95 (s, 1 H), 3.78 (s, 1 H), 3.35 (m, 1 H) 3.21 (t, 2 H), 2.30-0.91 (m, 34 H), 0.70 (s, 3 H). A solution was then made from 0.200 g (0.40 mmol) of the above conjugate, along with 1 mL of DMF, 0.2 mL of DIPEA, and O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)N,N,N′N′-tetramethyluronium tetrafluoroborate (0.152 g, 0.44 mmol), which was then stirred at room temperature for 2 h. To this solution was added, dropwise, a solution made from spermine (17.0 mg, 0.084 mmol) and 0.3 mL of DIPEA in 1 mL of DMF, and the resulting solution was stirred at 55 °C for 16 h. Removal of solvent under reduced pressure (50 °C) afforded a residue that was dissolved in 1 mL of methanol. Subsequent precipitation via the addition of 15 mL of a 10% aqueous NaHCO3 solution, followed by filtration and purification via preparative thin-layer chromatography (SiO2, CHCl3/MeOH/ H2O, 120/73/2, v/v/v), afforded 60 mg (35%) of 3 having Rf 0.50 (SiO2, CHCl3/MeOH/H2O, 120/73/2, v/v/v) and 1H NMR (CD3OD, 500 MHz) δ (ppm): 3.95 (br s, 4 H), 3.79 (m, 4 H), 3.35 (m, 4 H), 3.30-3.20 (m, 8 H), 3.18 (m, 12 H), 2.21-0.91(m, 144 H), 0.71 (s, 12 H). HRMS for C122H206N8O20 (Na+) Calcd: 2126.5241. Found: 2126.5343. Conjugate 4. A solution was prepared from cholic acid succinimide ester (1.015 g, 2 mmol), 7 mL of DMF, 1 mL of DIPEA, and sarcosine (0.178 g, 2 mmol) and allowed to stir at 50 °C for 48 h. The solvent was then removed under reduced pressure, and the residue purified by column chromatography (SiO2, CHCl3/MeOH/H2O, 120/ 100/5, v/v/v) to give 0.450 g (45%) of the corresponding sarcosine conjugate of cholic acid having Rf 0.54 (SiO2, CHCl3/CH3OH/H2O, 120/100/5, v/v/v) and 1H NMR (D2O, 500 MHz): δ (ppm): 4.0-3.94 (m, 3 H); 3.79 (s, 1 H), 3.41 (m, 1 H), 2.90 (d, 3 H), 2.31-0.91 (m, 30 H), 0.64 (s, 3 H). The above conjugate (0.100 g, 0.22 mmol) was dissolved in 0.2 mL of DIPEA plus 1 mL of DMF, followed by addition of O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)N,N,N′N′-tetramethyluronium tetrafluoroborate, (0.079 g, 0.23 mmol). This solution was then stirred at room temperature for 2 h, followed by the dropwise addition of a solution made from spermine (8.6 mg, 0.042 mmol)

1316 Bioconjugate Chem., Vol. 13, No. 6, 2002

and 0.2 mL of DIPEA in 0.5 mL of DMF; stirring was continued for an additional 16 h. Removal of solvent under reduced pressure (50 °C) afforded a residue that was dissolved in a minimum volume of methanol and added, dropwise, to a stirred 10% aqueous NaHCO3 solution (20 mL). The resulting solid was purified by column chromatography (SiO2, CHCl3/MeOH/H2O, 120/ 80/3, v/v/v) to give 36 mg (45%) of 4 having Rf 0.65 (SiO2, CHCl3/MeOH/H2O, 120/80/3, v/v/v) and 1H NMR [(CD3)2SO, 500 MHz] δ ppm 4.15 (s, 4 H), 3.94 (s, 4 H); 3.83 (s, 4 H), 3.69 (s, 4 H), 3.32-3.13 (m, 16 H), 2.95 (br s, 12 H),2.41-0.91(m,128H),0.70(s,12H).HRMS: C118H198N8O20(Na+) Calcd: 2070.4615. Found: 2070.4578. Conjugate 5. A suspension was prepared from cholic acid succinimide ester (0.566 g, 1.12 mmol), 1 mL of DIPEA, lysine (0.078 g, 0.5 mmol), and Na2CO3 (0.056 g, 0.5 mmol) in DMF and stirred for 16 h at 50 °C. Removal of solvent under reduced pressure (50 °C) afforded a residue that was dissolved in 1 mL of methanol. This methanolic solution was then added, dropwise, to a saturated aqueous Na2CO3 solution with stirring. The precipitate was removed by filtration and purified by column chromatography (SiO2,CHCl3/MeOH/H2O, 65/25/ 4, v/v/v) to give 0.356 g (72%) of the corresponding lysine conjugate of cholic acid having Rf 0.30 (SiO2,CHCl3/ MeOH/H2O, 65/25/4, v/v/v) and 1H NMR (CD3OD, 360 MHz) δ ppm 4.25 (m, 1 H), 3.94 (m, 2 H), 3.79 (s, 2 H), 3.35 (m, 2 H); 3.16 (t, 2 H), 2.21-0.91 (m, 66 H), 0.70 (s, 6 H). The above conjugate (0.100 g, 0.107 mmol) was dissolved in 0.2 mL of DIPEA plus 1 mL of DMF, followed by the addition of N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium terafluoroborate (0.039 g, 0.129 mmmol). The resulting solution was then allowed to stir for 2 h at 50 °C. To this solution was added a solution that was made from putrescine (0.3 mL), 0.2 mL of DIPEA, and 0.5 mL of DMF. After stirring for 16 h at 50 °C, the solvent was then removed under reduced pressure (50 °C) and the residue dissolved in 1 mL of methanol. This solution was then added, dropwise, to an 10% aqueous Na2CO3 solution. The resulting solid was collected by filtration and purified by column chromatography (SiO2, CHCl3/MeOH/H2O, 120/50/2, v/v/v) to give 30 mg (47%) of 5 having Rf 0.55 (SiO2, CHCl3/MeOH/H2O, 120/50/2, v/v/v), and 1H NMR [(CD3OD), 360 MHz]: δ (ppm): 4.25 (m, 2 H), 3.94 (m, 4 H), 3.79 (s, 4 H), 3.35 (m, 4 H), 3.16 (t, 8 H), 2.21-0.91 (m, 136 H), 0.70 (s, 12 H). HRMS for C122H188N6O18(Na+) Calcd: 1928.3872. Found: 1928.3809. Conjugate 6. To a solution of 5β-cholanic acid-3R,7Rdiol (0.106 g, 0.26 mmol) in 0.2 mL of DIPEA plus 1 mL of DMF was added O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′N′-tetramethyluronium tetrafluoroborate, (0.096 g, 0.28 mmol). After stirring for 2 h at room temperature, a solution of spermine (12.0 mg, 0.060 mmol) in 0.2 mL of DIPEA plus 0.5 mL of DMF was then added, dropwise, and the mixture was allowed to stir for an additional 16 h at room temperature. Removal of solvent under reduced pressure (50 °C) afforded a residue that was dissolved in 1 mL of methanol. Subsequent precipitation via addition to a saturated aqueous NaHCO3 solution, followed by filtration and purification by preparative thin-layer chromatography (SiO2, CHCl3/ MeOH/H2O, 100/20/1, v/v/v), afforded 50 mg (50%) of 6 having Rf 0.45 (SiO2, CHCl3/MeOH/H2O, 100/20/1, v/v/v) and 1H NMR (CD3OD 360 MHz) δ (ppm): 3.79 (bs, 4 H), 3.35 (m, 4 H), 3.22-3.36 (m, 8 H), 3.16 (m, 4 H), 2.210.91 (m, 136 H), 0.70 (s, 12 H). HRMS for C106H178N4O12(Na+) Calcd: 1722.3334. Found: 1722.3235.

Bandyopadhyay et al.

Conjugate 7. To a solution of 5β-cholanic acid-3R,12Rdiol (0.106 g, 0.27 mmol) in 0.2 mL of DIPEA plus 1 mL DMF was added O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin3-yl)-N,N,N′N′-tetramethyluronium tetrafluoroborate, (0.096 g, 0.28 mmol). After stirring the mixture at room temperature for 2 h, a solution of spermine (12.0 mg, 0.060 mmol) and 0.2 mL of DIPEA plus 0.5 mL of DMF was added, dropwise, and the mixture was allowed to stir for an additional 16 h at room temperature. Removal of solvent under reduced pressure (50 °C) afforded a residue that was dissolved in 1 mL of methanol. Subsequent precipitation via addition to an aqueous solution that was saturated with NaHCO3, and purification by preparative thin-layer chromatography (SiO2, CHCl3/MeOH/H2O, 100/20/1, v/v/v), afforded 55 mg (55%) of 7 having Rf 0.50 (SiO2, CHCl3/MeOH/H2O, 100/20/1, v/v/v) and 1H NMR (CD3OD, 360 MHz) δ (ppm): 3.90 (bs, 4 H), 3.35 (m, 4 H), 3.22-3.36 (m, 8 H), 3.16 (m, 4 H), 2.21-0.91 (m, 136 H), 0.70 (s, 12 H). HRMS for C106H178N4O12(Na+) Calcd: 1722.3334. Found: 1722.3298. RESULTS AND DISCUSSION

Ion Conductors from Biogenic Precursors. The specific molecules that were chosen as synthetic targets for this work were 2, 3, 4, 5, 6, and 7. Conjugates 2 and 3, bearing sterols derived from glycocholic acid and a

conjugate of cholic acid plus γ-amino butyric acid, respectively, extend the distance between each facial amphiphile and the spermine backbone, relative to 1; at the same time, they add four secondary amide groups, each of which may act as a hydrogen bond donor or acceptors a feature that could promote aggregation of the ion conductor, leading to increased activity. Ion conductor 4, which bears facial amphiphiles made from a conjugate of sarcosine and cholic acid, is an analogue of 2. In this case, each of the four additional amide groups can only function as hydrogen bond acceptors. Conjugate 5, de-

rived from four molecules of cholic acid, two lysine molecules, and one putrescine unit, may be viewed as an “expanded” analogue of 1, which also has additional space between each of the sterol units. In addition, this conjugate contains a total of six secondary amide groups.

Ion Conductors Derived from Biogenic Amines

Bioconjugate Chem., Vol. 13, No. 6, 2002 1317

Scheme 2

Scheme 3

Finally, conjugates 6 and 7 are close analogues of 1

having increased hydrophobicity; in this case, the hydroxyl group at the C12 and C7 carbons have been replaced by hydrogen atoms, respectively. Conjugate 2 was synthesized by direct condensation of glycocholic acid with spermine (not shown). Conjugate 3 was prepared by first coupling γ-amino butryic acid to the cholic acid framework (Scheme 2); conjugate 4 was prepared similarly, using sarcosine in place of γ-amino butryic acid. Acylation of both amino groups of lysine with an activated form of cholic acid, followed by condensation of the resulting carboxylic acid with putrescine afforded 5 (Scheme 3). Finally, conjugates 6 and 7 were obtained by direct condensation of spermine with 3R,7Rdihydroxycholic acid and 3R,12R-dihydroxycholic acid, respectively (not shown). Ion Conducting Properties. Liposomes that were used as model membranes in this work were in the form of 200 nm diameter unilamellar vesicles. Under the conditions used (i.e., 35 °C), liposomes derived from C14, as well as from C16, were maintained in the physiologically relevant fluid phase. Using procedures similar to those that have been described in detail elsewhere, the rate at which Na+ crosses these liposomal membranes was measured via 23 Na+ NMR spectroscopy (1l, 4). In all cases, varying percentages of ion conductor were introduced into the liposomes at the time of their preparation (i.e., “doublesided addition”). For these experiments, a membraneimpermeable paramagnetic shift reagent was added to the external aqueous phase so that liposomal-entrapped

and external Na+ could be distinguished. In all cases, pseudo-first-order rate constants (kobsd) exhibited a secondorder dependency on the mol % of the conjugate that was present. For one such experiment, where vesicles were made from C16, and 2 was used as an ion conductor, both the rate of 23Na+ influx and the rate of 7Li+ efflux were monitored, simultaneously. Within experimental error, the rate constants for both transport processes were identical. This finding lends support for an antiport mechanism of transport. Figure 1 shows typical plots for kobsd versus (mol % of 2) (2) for the two different membranes investigated (C14 and C16). As discussed previously, the linearity of such plots indicates that transport-active dimers are involved (1g). Here, it is assumed that only a small fraction of the conjugate is aggregated, where it can be shown that:

kobsd ) k2[monomer]2/K where K is the equilibrium constant for dissociation of the dimer, k2 is the rate constant for ion transport, and

Figure 1. Plot of kobsd versus (mol % 2)2 for vesicles made from (9) C14 and (b) C16 at 35 °C; inset shows data for C14 with an expanded x-axis.

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Bandyopadhyay et al.

Table 1. Ion Conducting Activities and Membrane Selectivitiesa ion conductor

lipid

104 k2/K (min-1 mol %-2)

S (C14/C16)

1

C14 C16 C14 C16 C14 C16 C14 C16 C14 C16 C14 C16 C14 C16

190000 130 8000 28 54000 56 29200 39 35000 15.5 10560 35.6 6028 23

1500

2 3 4 5 6 7

290 965 750 2258

lar hydrogen bonds in conformation B. Alternatively, the larger number of hydroxyl groups of 1 could increase the extent of hydration of the pathway for diffusion, which, in turn, could facilitate the flow of ions; that is, its intrinsic activity could be higher. Depsite these uncertainties, the results reported herein demonstrate the feasibility of constructing an entire family of ion conductors that span a moderate range of ion transport properties, based on biogenic precursors. Efforts aimed at extending this approach with a view toward drug design are continuing in our laboratories.

297

ACKNOWLEDGMENT

262

We are grateful to the National Science Foundation (Grant CHE-9612702) for support of this research.

a All kinetic experiments were carried out at 35 °C; the error in kobsd is estimated to be (10%.

[monomer] is the analytical concentration of the ion conductor that is present in the dispersion. Specific values of k2/K that have been determined for 2, 3, 4, 5, 6, and 7 are shown in Table 1. Since k2 and K cannot be separated by such analysis, we are limited to operational comparisons among the conjugates. Also listed in Table 1 are membrane-selectivity factors, S, which represent the ratio of (k2/K)14/(k2/K)16, where 14 and 16 refer to liposomes derived from C14 and C16, respectively. In all cases, significant ion conductivity was observed. When the thicker C16 membranes were used as model systems, the differences in ion conducting activity were modest. Thus, the conjugate having the highest activity (3) and the one having the lowest activity (5), within the series 2-7, differed by a factor of ca. 4. In addition, conjugate 1 was only ca. two times more active than 3. In the thinner C14 membranes, however, the differences in activity were more pronounced. In this case, 3, having the greatest activity, was ca. nine times more active than 7. A greater difference in activity within the thinner membrane was also apparent when 1 was compared to analogues 6 and 7. Here, removal of a single hydroxyl group from each of the sterols resulted in a reduction in Na+ transport activity by factors of 18 and 32, respectively. An examination of the S factors in Table 1 further reveals that membrane selectivities range from 262 for 7 to 2258 for 5. Thus, both the activity and membrane selectivity show a significant dependency on the structure and the composition of the ion conductor. In a broader context, the present findings demonstrate the generality of our design principle. Thus, all members within this family of conjugates derived from spermine, putrescine, lysine, γ-aminobutyric acid, sarcosine, cholic acid, glycocholic acid, 3R,7R-dihydroxycholic acid, and 3R,12R-dihydroxycholic acid showed significant activity in promoting the transport of Na+ across phospholipid membranes. Whether the observed variations in activity and membrane selectivity are due to differences in intrinsic activities (i.e., k2) or to the extent of aggregation (K) remains to be established. In addition, the extent to which each of these conjugates is drawn into the membrane via conformation B (Scheme 1) is uncertain. Thus, one can rationalize the greater activity of 1, relative to 6 and 7 in terms of a greater percentage of 1 being drawn into the bilayer through a larger number of intramolecu-

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