Biogenic Amine-Ionophore Interactions

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Proc. Nati. Acad. Scl. USA Vol. 74, No. 11, pp. 4734-4738, November 1977 Chemistry Biogenic amine-ionophore interactions: Structure and dynamics of lasalocid (X537A) complexes with phenethylamines and catecholamines in nonpolar solution (nuclear magnetic resonance/solution conformation/exchange kinetics) CYNTHIA SHEN AND DINSHAW J. PATEL Bell Laboratories, Murray Hill, New Jersey 07974 Communicated by F. A. Bovey, August 12,1977

ABSTRACT The ionophore lasalocid A forms 1:1 complexes alkaline earth ions are sandwiched between the polar faces of with phenethylamines (1-amino-1-phenylethane and 1-amino- two lasalocid anions in nonpolar solution (3, 12, 13) and in 2-phenylethane) and catecholamines (dopamine and norepinephrine) in nonpolar solution. We have undertaken high-res- crystals grown from the same medium (7-10). By contrast, olution proton nuclear magnetic resonance studies to deduce monomeric structures, in which the metal ion chelates to the structural and kinetic information on the ionophore-biogenic polar face of one lasalocid anion and solvent, are observed in amine complexes in chloroform solution. The coupling constant, polar media (4, 14). chemical shift, and relaxation time data demonstrate that the A number of biological studies have implicated the ability lasalocid backbone conformation and the primary amine of lasalocid to affect the distribution of biogenic amines across binding sites in the complexes are similar to those determined earlier for the alkali and alkaline earth complexes of this iono- the membrane (18-21). Westley and coworkers have demon- phore in solution. The exchange of lasalocid between the free strated that lasalocid A forms crystalline complexes with pri- acid (HX) and the primary amine complexes (RNH3X) in chlo- mary biogenic amines (22) and this has led us to undertake roform solution have been evaluated from the temperature- structural and kinetic investigations of these complexes in so- dependent line shapes at superconducting fields. The kinetic lution. These amines include 2-aminoheptane (1), the phen- parameters associated with the unimolecular dissociation ethylamines [1-amino-l-phenylethane (2), and 1-amino-2- phenylethane (3a)], and the catecholamines [dopamine (3b) and (RNH3X T RNH2 + HX) norepinephrine (3c)] (Fig. 2). and the bimolecular exchange (RNH3X + HX* k2 RNH3X* + HX) EXPERIMENTAL Materials. Lasalocid (ethanol-free) and R(+)- and S(-)- reactions have been deduced from an analysis of the lifetime 1-amino-l-phenylethanes were generous gifts from J. W. of the complex as a function of the reactant concentrations. The relative stability of the complex decreases in the order phenyl Westley and R. Evans, Jr., of Hoffman-La Roche, Nutley, NJ. > n-pentyl for substituents on the carbon a to the amino group Dopamine and R(+)-norepinephrine were purchased as their (1-amino-l-phenylethane and 2-aminoheptane) and phenyl > hydrochloride salts from Norse Laboratories, Santa Barbara, 3,4-dihydroxyphenyl for substituents on the carbon # to the CA. Deuterated chloroform, deuterated methylene chloride, amino group (l-amino-2-phenylethane and dopamine). These and 1-amino-2-phenylethane were purchased from Aldrich results suggest that nonpolar interactions between the biogenic Chemical Co. The solvents were dried over molecular sieves amine side chain and the lasalocid molecule contribute to the prior to use. stability of the complex in solution. Methods. Proton nuclear magnetic resonance (NMR) spectra Lasalocid A [see Fig. 1, for chemical sequence (1) and revised (360 MHz) were obtained in the Fourier transform mode on numbering system (2)] belongs to the family of linear carboxylic a Bruker HX-360 spectrometer interfaced with a Nicolet polyether antibiotics that transport alkali ions across membranes BNC-12 computer system. Proton longitudinal relaxation times (5, 6). The backbone of these ionophores adopts a folded (T1) were measured by using the (ir,r, ir/2) pulse sequence. head-to-tail conformation stabilized by intramolecular hy- The amine-lasalocid complexes of 1, 2, and 3a were gener- drogen bonds between the carboxylic and hydroxyl groups (refs. ated by mixing equimolar ratios of the amine and lasalocid in 5 and 6 and the references therein). The ionophores complex methylene chloride solution, followed by gradual addition of the alkali ions through their hydroxyl, ether, carbonyl, and n-hexane to precipitate the complexes. We followed the pro- carboxylate groups, resulting in a hydrophobic exterior which cedure of Westley et al. (22) to generate the lasalocid complexes facilitates the transport of metal ions across membranes. The of dopamine (3b) and R(+)-norepinephrine (3c). conformation of lasalocid resembles a flat disc with a polar and a nonpolar face (7-10), and it differs from the other carboxylic RESULTS AND DISCUSSION polyether antibiotics of larger dimensions which can form polar Stoichiometry. We have monitored the interaction of lasa- cavities for metal ion coordination. This permits the polar face locid with biogenic amines by following the chemical shift of lasalocid to coordinate ions with different radii and charges, changes of the ionophore proton NMR resonances on addition including alkali, alkaline earth (3, 4, 11-14), rare earth (15), and of amines to saturation concentrations. The exchange rate be- transition (16) metal ions as well as amines (17). The alkali and tween the free and complexed states (as monitored at the H5, The costs of publication of this article were defrayed in part by the H6, and HI1 resonances) was slow on the NMR time scale for payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: NMR, nuclear magnetic resonance; T1, proton longi- this fact. tudinal relaxation times. 4734 Downloaded by guest on October 1, 2021 Chemistry: Shen and Patel Proc. Natl. Acad. Sci. USA 74 (1977) 4735

.CH 3, [4] L5J L,/ J CH3 24 CH3 FIG. 1. Formula of lasalocid A. We have adopted the recently proposed numbering system (2), which differs from that was used in our previous papers (3, 4). from a comparison of the relative areas for a given resonance and at H5, H6, and H23 (located on the periphery of the folded in each state. By contrast, fast exchange between free and ionophore structure) (Table 2). complexed states was observed for amines 3b and 3c so that the The lasalocid H8 and HI, protons located on the polar face stoichiometry of the complex could be evaluated from the av- of the ionophore exhibited the largest decrease in T1 values on erage chemical shift changes for a given resonance of lasalocid complex formation (Table 2). This suggests that the biogenic on the gradual addition of amine (23). These studies establish amine binds to the polar face as observed in the crystalline state the formation of 1:1 complexes between lasalocid and the pri- (22), and the short T1 values for H8 and HI1 in the complexes mary amines 1, 2, 3a, 3b, and 3c in chloroform solution. The reflect proton-proton dipolar contributions to the relaxation complexation shifts are summarized in Table 1. time from proximal biogenic amine protons. Molecular Dimensions. X-ray studies have demonstrated We have also evaluated T1 for the biogenic amine protons that lasalocid forms 1:1 monomeric complexes with 1-amino- in these complexes and found them to be much shorter than the 1-phenylethane (2) for crystals grown from nonpolar solvents corresponding values expected for the free amines in solution. (22). This is in contrast to the dimeric structures (Na2X2, BaX2) For example, the relaxation times are 0.59 sec (NHS+ protons), observed in the crystalline state with the less bulky alkali and 0.54 ± 0.02 sec (CaH proton), 0.40 sec (CaCH3 protons), and alkaline earth ions (4, 15). The 360-MHz T1 values for lasalocid 1.1 + 0.2 sec (aromatic protons) in the 1-amino-1-phenyleth- A and its complexes with 2, 3a, 3b, and 3c in chloroform solu- ane-lasalocid A complex in chloroform solution at 20°. tion are summarized in Table 2. Structural Aspects. The x-ray structure of the 1:1 complex Previous studies have established a monomeric structure for of R(+)-l-amino-l-p-bromophenylethane and lasalocid es- lasalocid in nonpolar solution (3) so that the molecular weight tablishes that the amine nitrogen coordinates the ionophore by increases from 590 for the free acid to 711 for its complexes with hydrogen bonding at carboxylate 02, ether 06, and hydroxyl 2 and 3a and to 750 10 for its complexes with 3b and 3c. The 08 located on the polar face of the folded backbone of lasalocid increased value of the rotational correlation time in the 1:1 in the crystalline state (22). complexes should result in shorter T1 values compared to the The vicinal proton-proton coupling constants across C10-C11, values for the free acid. This was observed for the ionophore C11-C12, C14-CI5, and C15-CI6 single bonds were evaluated resonances at H12, H14, and H19 (located on the nonpolar face) for the 1:1 biogenic amine-lasalocid complexes and were similar

Table 1. Lasalocid proton chemical shift changes upon complex formation with biogenic amines in chloroform at 270* HX-RNH3X H5 H6 H8 Hi, H12 H14 H15 Hi9 H23 For RNH2 1 -0.15 -0.14 +0.82 +0.22 -0.08 -0.14 +0.27 -0.03 -0.19 2a -0.13 -0.12 +0.81 +0.27 -0.09 -0.17 +0.21 -0.15 -0.40 3a -0.15 -0.14 +0.79 +0.19 -0.09 -0.14 +0.25 -0.03 -0.28 3b -0.13 -0.11 +0.57 +0.20 +0.01 -0.14 +0.13 -0.07 -0.59 3c -0.11 -0.09 +0.69 +0.31 +0.01 -0.10 +0.16 -0.03 -0.27 HXt 7.15 6.61 3.32 4.08 2.81 2.60 3.87 3.47 3.95 * (-) For upfield shifts, (+) for downfield shifts. The concentration of all species was -10 mM. t Chemical shifts of ethanol-free lasalocid A(HX) protons.

Table 2. Proton relaxation times (T1 in sec) in chloroform at 200* RNH3X H5 H6 H8 Hi, H12 H14 Hi9 H23 RNH2 2 0.22 0.33 0.51 0.45 0.50 0.60 3a 1.17 1.04 0.26 0.37 0.38 0.50 0.51 0.85 3b 1.14 0.94 0.24 0.35 - 0.48 0.79 3c 1.12 0.78 - 0.38 0.41 0.46 0.60 HX 1.53 0.95 0.35 0.60 0.51 0.60 0.61 * The concentration of all species was -5.5 mM; 360 MHz, 900 pulse width = 14.5 ,usec; repetition time = 6 sec. Downloaded by guest on October 1, 2021 4736 Chemistry: Shen and Patel Proc. Natl. Acad. Sci. USA 74 (1977) * CH3 CH2CH2 CH2 CH2 - CH - NHz Temperature, 0C 60 40 20 0 40 20 0 -20 [1] ICH 103 103 CH-2NH2 ~~~~~~~~~~~~~I[2] . 102 R2 CH-CH21-NH "I-,- 102

R2 [3] (3a) R1 = R2= H 101 101 (3b) R1 = H, R2= OH 3.1 3.3 3.5 3.7 3.3 3.5 3.7 3.9 (3c) R1 = R2= OH 1/temperature, X 10' K` FIG. 2. Structures of 2-aminoheptane [1], 1-amino-1-phenyl- FIG. 4. Semilogarithmic plots of TC-1 versus reciprocal of tem- ethane [21, 1-amino-2-phenylethane [3a], dopamine [3b], and nor- perature for the exchange between equimolar concentrations of HX epinephrine [3c]. and (Left) its complex with 1-amino-1-phenylethane (2) at 12.0 mM (A), 67.8 mM (O), and 122.5 mM (0) and (Right) its complex with within +1 Hz to the corresponding values in the free acid and norepinephrine (3c) at 2.5 mM (A), 10.0 mM (O), and 20.0 mM alkali and alkaline earth complexes (3, 4, 24). This demonstrates (0). that the ionophore backbone conformation extending from C1o with the most pronounced change observed for 1-amino-i- to C16 is similar for lasalocid in both the absence and the pres- phenylethane (2) and dopamine (3b). ence of complexing metal ions and biogenic amines in nonpolar Exchange Parameters. We monitored the exchange between solvents, in agreement with related crystallographic results the free acid (HX) and the complexes (RNH3X) of the amines (7-10, 22). (RNH2) la, 2, 3a, 3b, and 3c in chloroform solution. The ex- We observed downfield shifts for those lasalocid protons on perimental line shapes of the H5, H6, and H11 resonances for the polar face in close proximity to the biogenic amine binding equimolar ratios of HX and RNH3X were analyzed by using site observed in the crystalline state (22). These included reso- the DNMR 2 program of Binsch and Kleier to on (25) deduce the nances H8 and H11 the polar face and H15 located just below lifetime in the complex state, rc as a function of temperature. the oxygen cluster made up of ether and hydroxyl groups (Table The results are presented in Fig. 3. 1). The resonances H12, H14, and H19 located on the nonpolar The exchange between HX and RNH3X is slow enough to face exhibited small upfield shifts on complex formation be measured above room temperature for amines 1 and 2, whereas the upfield shifts at H5 and H6 reflect the ionization which have n-pentyl and phenyl groups, respectively, attached of to the anion in The the free acid lasalocid the complex (3, 4). to the carbon a to the amino group. We observed a larger i-c-1 H23 resonance shifted upfield in the biogenic amine complexes, value for the complex with 1 compared to 2, indicative of the greater stabilization of the complex by the phenyl ring com- Temperature, °C pared to the n-pentyl side chain (Fig. 3 left). We also evaluated 60 40 20 0 40 20 0 -20 and compared the TC-1 values of the complexes with amines 3a, 3b, and 3c in exchange with equimolar free HX in chloroform solution (Fig. 3 right). The presence of the hydroxyl 103 groups on dopamine (3b) and norepinephrine (3c) destabilizes their respective complexes. These results suggest that nonpolar interactions between the biogenic amine side chain and the lasalocid molecule contribute to the stability of the complex in T solution. This conclusion receives further support from our 102 observation that the lifetime of the lasalocid-ammonium complex (<1 msec) is much shorter than the lifetimes observed I.. for the lasalocid-biogenic amine complexes (10-100 msec) in chloroform at 0°. The exchange between the free acid and the 1-amino-i- phenylethane complex in chloroform solution was studied as a function of reactant concentrations to elucidate the details of 101 . I I I I , _II 3.1 3.3 3.5 3.1 3.3 3.5 3.7 3.9 4.1 the exchange process. The rc-1 values were independent of the 1/temperature, X 103 K-1 concentration of the complex but dependent on the free acid concentration as determined from experiments in which one FIG. 3. Semilogarithmic plots of r, -1 versus reciprocal of temperature for the exchange between HX (12 mM) and its complexes of the reactant concentrations was fixed while the other con- (12 mM) with (Left) 1 (0) and 2 (A) and (Right) with 3a (O), 3b (A), centration was varied. 1 and 3c (0). The TC-1 values are calculated from a line shape analysis The ic- values for equimolar population of reactants are of H6 for 1, H5, H6, and H1, for 2 and 3a, and Hs for 3b and 3c. plotted as a function of temperature in Fig. 4 left. We did not Downloaded by guest on October 1, 2021 Chemistry: Shen and Patel Proc. Natl. Acad. Sci. USA 74 (1977) 4737

Temperature, 0C centrations. The dissociation rate constant of the norepinephrine 50 40 30 20 I0 complex (430 sec' at 250) is much higher than that estimated for the 1-amino-l-phenylethane complex (24 sect at 250). This clearly demonstrates the destabilizing effect of the hydroxyl substituents on the amine-lasalocid complexes. The activation parameters for dissociation of the norepinephrine complex are AH* = 6.8 kcal/mol and AS* = -23.8 eu in chloroform at 25°. sec Enantioselectivity. There has been considerable interest in the optical resolution of organic compounds by complex formation with chiral cyclic polyethers (26, 27), cyclodextrins (28, 29), and ion-binding cyclic hexapeptides (30). The C"H and CcCH3 protons of the R(+) and S(-) isomers of I-amino-l-p-bromophenylethane exhibit different chemical c shifts in their complexes with lasalocid in chloroform solution

c'U [4.67 ppm, 1.65 ppm for the R(+) complex compared to 4.56 0 ppm, 1.72 ppm for the S(-) complex]. The chemical shift dif- a' ferences indicate that the H and CH3 groups attached to the asymmetric carbon of the amine occupy somewhat different environments in the two complexes. We monitored the exchange between the free acid and optically pure R(+) and S(-) complexes of I-amino-l-p-bromophenylethane and deduced the r,-1 values as a function of temperature in chloroform solution. The rate data were identical for the two enantiomers within experimental error, suggesting that the two diastereomeric complexes exhibit similar stabilities in chloroform solution. Westley and coworkers (22) demonstrated that the R(+) configuration of 1-amino-l-phenylethane preferentially co- 101 crystallizes with lasalocid from a mixture of the racemic amine and the ionophore. This suggests that solubility differences 3.0 3.2 3.4 3.6 between the two diastereomeric complexes make the pre- 1/temperature, X 103 K-l dominant contribution to the optical resolution of asymmetric FIG. 5. Plot of estimated first-order dissociati4 on rate constant amines by preferential crystallization as lasalocid salts. ki and bimolecular exchange rate constant k2 versus reciprocal of temperature for exchange between HX and equal population of its We thank Dr. J. W. Westley, Dr. J. F. Blount, and Mr. R. H. Evans, complex with 1-amino-1-phenylethane (see legend of Fig. 4). Jr., of Hoffmann-La Roche, Nutley, NJ, and Prof. S. R. Simon of State University of New York at Stony Brook for many stimulating discus- sions. C.S. was supported under National Institutes of Health Grant observe a 1:1 correspondence between an increase in the HX HL-16474 (principal investigators, Profs. S. R. Simon and H. L. concentration and a corresponding increase in the m'_- value Friedman). as would be expected for a sole bimolecular exchange process: 1. Westley, J. W., Evans, R. H., Jr., Williams, T. & Stempel, A. (1970) Chem. Commun., 71-72. k2 + HX* + 2. Westley, J. W. (1976) J. Antiblot. 29,584-586. RNH3X Z RNH3X* HX. 3. Patel, D. J. & Shen, C. (1976) Proc. Natl. Acad. Sci. USA 73, The dissociation of the complex into neutral amine and free acid 1786-1790. in nonpolar solution could provide an additional pathway for 4. Shen, C. & Patel, D. J. (1976) Proc. Nati. Acad. Sci. USA 73, exchange 4277-4281. k1 5. Ovchinnikov, Yu. A., Ivanov, V. T. & Shkrob, A. M. (1974) RNH3X RNH2 + HX. Membrane Active Complexones (Elsevier Scientific Publishing +. Co., New York), pp. 202-205. The TCr1 value for the proposed mechanism that incorporates 6. Pressman, B. C. (1976) Annu. Rev. Biochem. 45,501-530. 7. Bissell, E. C. & Paul, I. C. (1972) Chem. Commun., 967-968. both reactions as contributors to the exchange process is given 8. Johnson, S. M., Herrin, J., Liu, S. J. & Paul, I. C. (1970) J. Am. by rc-I = k2(HX) + ki. The rate constants ki and k2 can be Chem. Soc. 92,4428-4435. evaluated from a plot of Tr,1 versus HX concentration. 9. Schmidt, P. G., Wang, A. H. J. & Paul, I. C. (1974) J. Am. Chem. The temperature-dependence of the unimolecular rate Soc. 96,6189-6192. constant kI (24 sec1 at 250; AH* = 9.2 kcal/mol; AS* = -22.0 10. Maier, C. & Paul, I. C. (1971) Chem. Commun., 181-182. eu) and the bimolecular rate constant k2 (1100 M-1 sec-I at 250; 11. Pressman, B. C. (1973) Inorganic Biochemistry (Elsevier Sci- AH* = 9.7 kcal/mol; AS* = -12.0 eu) is summarized in Fig. entific Pub. Co., New York), Vol. 1, pp. 204-226. 5 for the exchange between the free acid and the 1-amino-i- 12. Degani, H. & Friedman, H. L. (1974) Biochemistry 13,5022- phenylethane complex in chloroform solution. 5031. 13. Alpha, S. R. & Brady, A. H. (1973) J. Am. Chem. Soc. 95, The TC-I values for the norepinephrine complex in exchange 7043-7049. with equimolar population of free acid was independent of the 14. Cornelius, G., Gartner, W. & Haynes, D. H. (1974) Biochemistry concentration of the reactants (Fig. 4 right). This demonstrates 13,3052-3057. that only the unimolecular dissociation of the norepinephrine 15. Fernandez, M. S., Celis, C. H. & Montal, M. (1973) Biochim. complex contributes to the exchange mechanism at these con- Blophys. Acta 323,600-605. Downloaded by guest on October 1, 2021 4738 Chemistry: Shen and Patel Proc. Nati. Acad. Sci. USA 74 (1977)

16. Degani, H. & Friedman, H. L. (1974) Biochemistry 14,3755- Resolution Nuclear Magnetic Resonance (McGraw-Hill, New 3761. York), pp. 201-207. 17. Pressman, B. C. (1972) in The Role of Membranes in Metabolic 24. Anteunis, M. J. 0. (1976) Bioorgan. Chem. 5,327-337. Regulation, eds. Melman, M. A. & Hanson, R. W. (Academic 25. Binsch, G. & Kleier, D. A. (1969) Quantum Chemistry Program Press, New York), pp. 149-164. Exchange, No. 140 (Indiana University, Chemistry Depart- 18. Foreman, J. C., Mongar, J. L. & Gomperts, B. D. (1973) Nature ment). 245,249-251. 26. Chao, Y. & Cram, D. J. (1976) J. Am. Chem. Soc. 98, 1015- 19. Nordmann, J. J. & Cunnell, G. A. (1975) Nature 253, 646- 1017. 647. 27. Kyba, E. A., Koga, K., Sonsa, L. R., Siegel, M. G. & Cram, D. J. 20. Pasantes-Morales, H., Salced, R. & Gomez-Puyou, A. (1974) (1973) J. Am. Chem. Soc. 95,2692-2693. Biochem. Biophys. Res. Commun. 28. Mikotajczyk, M. & Drabowicz, J. (1971) Chem. Commun., 58,847-853. 317-318. 21. Holz, R. W. (1974) Biochim. Biophys. Acta 375, 138-152. 29. Benschop, H. P. & Van der Berg, G. R. (1970) Chem. Commun., 22. Westley, J. W., Evans, R. H., Jr. & Blount, J. F. (1977) J. Am. 1431-1432. Chem. Soc. 99,6057-6061. 30. Deber, C. M. & Blout, E. R. (1974) J. Am. Chem. Soc. 96, 23. Pople, J. A., Schneider, W. G. & Berstein, H. J. (1959) High 7566-7568. Downloaded by guest on October 1, 2021