On Tetramethylammonium Ion Role in Glycodeoxycholate Micellar

strength was varied by adding tetramethylammonium chloride (TMACl), although ... Tetramethylammonium (TMAGDC) and sodium glycodeoxycholate. (NaGDC) ...
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On Tetramethylammonium Ion Role in Glycodeoxycholate Micellar Aggregate Formation Angelo Antonio D’Archivio,† Luciano Galantini,*,‡ Enrico Gavuzzo,§ Edoardo Giglio,‡ and Francesco Punzo‡ Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` di L’Aquila, 67010 Coppito Due, L’Aquila, Italy, Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, and Istituto di Strutturistica Chimica “Giordano Giacomello” CNR, CP No 10, 00016 Monterotondo Stazione, Roma, Italy Received December 5, 2000. In Final Form: February 19, 2001 Previously, fiber and crystal structural models were proposed for bile acid salt micellar aggregates and verified in aqueous solutions. Electromotive force (emf) measurements on sodium salts versus ionic strength, pH, and bile salt concentration provided micellar aggregate compositions that supported the models. Ionic strength was varied by adding tetramethylammonium chloride (TMACl), although tetramethylammonium (TMA+) and Na+ ions could interfere in the aggregate formation and structure. In this case emf results cannot be used for sodium salts. Tetramethylammonium (TMAGDC) and sodium glycodeoxycholate (NaGDC), which forms helical aggregates constituted by trimers, are studied to clarify this point. TMAGDC crystal structure is solved, and circular dichroism (CD), quasi-elastic light-scattering (QELS), electrolytic conductance, and dielectric measurements on TMAGDC and NaGDC aqueous solutions are compared for determining similarities and dissimilarities in their behavior. TMA+ and Na+ coordinations in TMAGDC and NaGDC crystals show that Na+ potential energy is lower than that of TMA+, thus suggesting a stronger Na+ binding to glycodeoxycholate anion (GDC-) aggregates. Bilirubin-IXR (BR) chiral recognition is sensitive to aggregate structures. BR CD spectra suggest similar structures for TMAGDC and NaGDC anion aggregates. QELS measurements indicate that GDC- aggregates have a greater affinity for Na+ ions than for TMA+ ions and that TMA+ ions form TMAGDC aggregates that are smaller than those formed by NaGDC and could interrupt NaGDC aggregate growth. From conductance data TMA+ seems to be bound to anion aggregates less than Na+, enhances its interactions when micellar size increases, could be included together with Cl-, coming from TMACl, into micellar aggregates, and could be bound through hydrophobic forces with the apolar lateral surface of anion aggregates. High TMAGDC values of the average electric dipole moment per monomer µ can be justified by cation and anion hydration. Probably, aggregate composition, population, and structure change slightly or do not change at all within the range 15-45 °C, where µ is nearly constant. The high single monomer µ (more than 70 D) suggests that TMA+ is anchored to GDC- in dilute solution, thus forming an ion pair. TMAGDC and NaGDC µ trends are both interpreted assuming a two-structure model and an equilibrium between dimeric and trimeric species. In conclusion, TMAGDC and NaGDC bigger aggregates have similar structures, even though the TMAGDC micellar size is smaller than that of NaGDC.

Introduction Transitions [aqueous micellar solution] f [gel] f [fiber] and, sometimes, [fiber] f [crystal] were observed for some dihydroxy and trihydroxy bile salts. Often, a fiber can be drawn from a gel or an aqueous micellar solution near the gelation point, and crystals can be obtained from a fiber by aging. Probably, the same structural unit, or a very similar one, could be present in fibers and crystals as well as in aqueous micellar solutions. Crystals and fibers were used as a source of models that were verified in aqueous micellar solutions. Of course, fiber models are more reliable than crystal ones, because the fiber is a system nearer to the aqueous micellar solution than is the crystal. Moreover, transitions [aqueous micellar solution] f [gel] f [fiber] may occur without drastic structural changes, since the gel is obtained concentrating the aqueous micellar solution and the fiber is drawn from the gel by means of the weak force of gravity. Models with well-defined packing structures, which suitably represent micellar aggregates, were obtained by * Corresponding author. E-mail: [email protected]. † Universita ` di L’Aquila. ‡ Dipartimento di Chimica, Universita ` di Roma “La Sapienza”. § Istituto di Strutturistica Chimica “Giordano Giacomello” CNR.

solving several crystal and fiber structures. Different models were observed for dihydroxy and trihydroxy bile salts in agreement with their very dissimilar physicochemical properties.1,2 Fibers of lithium, sodium, potassium, and rubidium deoxycholate (LiDC, NaDC, KDC, and RbDC, respectively)3,4 on the one hand, and those of sodium (NaGDC) and rubidium (RbGDC) glycodeoxycholate,5 sodium (NaTDC),5 rubidium (RbTDC),5 and calcium (CaTDC)6 taurodeoxycholate on the other, are formed by very similar 8/1 or 7/1 helices, respectively. A trimer with a 3-fold rotation axis is the repetitive unit (the basic building block) of both helices (Figure 1). These helices (see Figure 2 for 7/1 helix) were satisfactorily used for dihydroxy salt aggregate structures. Sodium taurocholate (1) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1; pp 249-356. (2) Carey, M. C. In Sterols and Bile Acids; Danielsson, H., Sjøvall, J., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1985; pp 345-403. (3) D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998, 14, 4776. (4) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. J. Phys. Chem. B 1999, 103, 4986. (5) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180. (6) D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 4197.

10.1021/la001704p CCC: $20.00 © 2001 American Chemical Society Published on Web 06/02/2001

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Figure 2. GDC- 7/1 helix projected along an axis perpendicular to the helical axis (top) and along the helical axis (bottom).

Figure 1. Atomic numbering of GDC- (top); GDC- trimer projection of a 7/1 helix along the helical axis (middle) and along an axis perpendicular to the helical axis (bottom). The thicker line represents an anion nearer to the observer.

(NaTC) and glycocholate (NaGC) crystals and fibers7-10 are constituted by dimers and octamers as basic units which suitably represent trihydroxy salt aggregate structures. Dihydroxy and trihydroxy salt structural units are mainly stabilized by strong polar interactions between counterions and anion aggregates. These models were verified by studying aqueous solutions of bile acid salts and of their interaction complexes with some probe molecules by means of commonly used and less conventional techniques in the micelle field. Measurements of small-angle X-ray scattering, electron spin and nuclear magnetic resonance, quasi-elastic lightscattering (QELS), and electrolytic conductance on the (7) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367. (8) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza L. Trans. Faraday Soc. 1994, 90, 1523. (9) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15, 2996. (10) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. Langmuir 2000, 16, 10436.

one hand, and extended X-ray absorption fine structure, electromotive force (emf), circular dichroism (CD), and dielectric spectroscopy on the other, were performed. In particular, emf measurements carried out by means of the constant ionic medium method as a function of ionic strength, pH, and bile salt concentration gave valuable information on micellar aggregate composition11-14 and confirmed the proposed models.3-5,9 However, ionic strength was varied in aqueous solutions by adding to sodium bile salts tetramethylammonium chloride (TMACl), although tetramethylammonium ion (TMA+) and Na+ could interfere in micellar aggregate formation, thus changing sodium salt structures. TMA+ influence on the formation and structure of glycodeoxycholate anion (GDC-) micellar aggregates in aqueous solutions and the effect of a possible competition between TMA+ and Na+ have been studied to establish whether emf data refer to anion aggregate structures similar to those of sodium salts. Tetramethylammonium glycodeoxycholate (TMAGDC) crystal structure has been solved to determine the nature and strength of TMA+‚‚‚ GDC- interactions and to compare them with those observed between Na+ and GDC- in a crystal structure previously solved.15 CD, QELS, conductance, and dielectric (11) Bottari, E.; Festa, M. R.; Jasionowska, R. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 443. (12) Bottari, E.; Festa, M. R. Mh. Chem. 1993, 124, 1119. (13) Bottari, E.; Festa, M. R. Langmuir 1996, 12, 1777. (14) Bottari, E.; Festa, M. R.; Franco, M. Analyst 1999, 124, 887. (15) Campanelli, A. R.; Candeloro De Sanctis, S.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536.

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measurements on TMAGDC and NaGDC aqueous micellar solutions have been accomplished for establishing similarities and dissimilarities in their behavior. Experimental Section Materials. TMAGDC, which was prepared from glycodeoxycholic acid (Sigma) and tetramethylammonium hydroxide (Carlo Erba), and NaGDC (Sigma) were twice crystallized from a mixture of water and acetone. Water and acetone were released from crystals by heating at 50 °C and under a pressure of 10-2 mmHg. NaCl was from Merck (suprapur). TMACl from Fluka (purum) was crystallized from a mixture of water and ethanol. BilirubinIXR (BR) puriss. (about 99%, Fluka) was used. High-performance liquid chromatography measurements showed that the commercial sample contains at least 96% BR. Trizma base 0.05 M, reagent grade, was from Sigma. X-ray Diffraction Measurements. X-ray data of a TMAGDC crystal were collected at room temperature, using Cu KR radiation (λ ) 1.5418 Å), by means of a Rigaku AFC5R diffractometer, equipped with a 12 kW rotating anode and a graphite monochromator. Cell constants and an orientation matrix were obtained from a least-squares fit of the angular settings of 25 carefully centered reflections in the range 25.0 e 2θ e 45.5°. Intensity data were collected by the ω-2θ mode at a variable and appropriate speed to a maximum 2θ of 124°. Stationary background counts were recorded on each side of the reflection. Peak counting was twice that of background. Refinement was carried out using 2212 reflections with I > 3σ(I). Intensities of three standard reflections, measured after every 147 reflections, were found to be practically constant throughout data collection, and therefore, no decay correction was applied. Data were corrected for Lorentz and polarization effects. No absorption correction was applied. The structure was solved by direct methods using the program SIR9216 and refined anisotropically by the full-matrix leastsquares method with the program SIR97.17 TMA+ carbon atoms were refined isotropically. The function minimized was ∑w(|Fo| - |Fc|)2, where Fo and Fc are observed and calculated structure factors, respectively, and w ) (sin θ/λ)2. Atomic scattering factors and anomalous dispersion coefficients were taken from ref 18. The correction for real and imaginary parts of anomalous dispersion was applied. Hydrogen atoms were generated at the expected positions, except water and hydroxyl hydrogens which were not considered in calculations. Their atomic coordinates were not refined, and their thermal parameters were set equal to the isotropic ones of carrier atoms. Final agreement factors R and Rw were 0.074 and 0.084, respectively. Bond distances and bond angles were in satisfactory agreement with those usually reported in the literature, except two N-C bond distances (1.30 and 1.57 Å) of TMA+. No unusual intermolecular contacts were observed. Listings of observed and calculated structure factors, atomic fractional coordinates together with equivalent isotropic thermal parameters, and bond lengths and bond angles are available as Supporting Information (see paragraph at the end of the paper). Density measurements were accomplished by flotation using a chloroform/n-hexane mixture, the density of which was determined by an Anton Paar DMA 02C densimeter. CD Measurements. CD spectra were recorded on a JASCO J-500A spectropolarimeter at 25 °C by using quartz cells with path lengths of 1.0 cm and by flushing with dry ultrapurified nitrogen before and during the experiments. A slit program that gives a wavelength accuracy better than 0.5 nm was used. The instrument was calibrated with androsterone (1.69 × 10-3 M in dioxane) on the basis of a molar ellipticity [θ]304 ) 11 180 deg cm2 dmol-1. All the CD spectra were recorded starting from 3 min of BR addition to 0.05 M NaGDC and TMAGDC aqueous solutions (16) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M. C.; Polidori, G.; Camalli, M. J. Appl. Crystallogr. 1994, 27, 435. (17) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Moliterno, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 1999, 32, 115. (18) International Tables for X-ray Crystallography; Kynoch Press: Birmingham, England, 1974; Vol. IV.

D’Archivio et al. and refer to a 1.0 cm path length. BR (4.6 × 10-6 M) was added by using a 0.01 M aqueous NaOH vehicle. QELS Measurements. A Brookhaven instrument consisting of a BI-2030AT digital correlator with 136 channels and a BI200SM goniometer was used. The light source was an argon ion laser, model 85, from Lexel Corporation operating at 514.5 nm. Dust was eliminated by means of a Brookhaven ultrafiltration unit (BIUU1) for flow-through cells, the volume of the flow cell being about 1.0 cm3. Nuclepore filters with a pore size of 0.1 µm were used. Samples were placed in the cell for at least 30 min prior to measurement to allow for thermal equilibration. Their temperature was kept constant at 25 °C within 0.5 °C by a circulating water bath. Measurements were repeated after 24 and 48 h to be sure that data were reproducible. Scattered intensity and time-dependent light scattering correlation functions were analyzed at a fixed angle of 90°. Scattered intensity and apparent diffusion coefficients did not depend on the exchanged wave vector in the range 30-150° in our experimental conditions. Scattering decays were analyzed by means of cumulant expansion up to second order, because higher order contributions did not improve the statistics. Results are reported in terms of the apparent hydrodynamic radius (Rh) obtained by the Stokes-Einstein relationship. Electrolytic Conductance Measurements. Impedance Analyzer HP 4194 A, which covers the frequency range 10 kHz to 100 MHz, was used. Impedance of the cell filled with the sample was measured in the narrow range of frequencies 10-100 kHz. Within this interval the phase angle is nearly zero, because impedance is the sample electric resistance and it is inversely proportional to its conductance. Frequencies in this interval are so much lower than those of solute and, even more so, of solvent dielectric relaxation, as to exclude any contribution of dielectric loss to measured resistance. This ensures that ionic or static conductance of the sample is measured. The cell constant was determined by measuring standard NaCl aqueous solutions. Conductivity measurements show standard deviations and reproducibility less than (2%. Dielectric Measurements. A model HP 4291 A impedance analyzer was used in the frequency range 1.0 MHz to 1.8 GHz. Measured reflection coefficients and phase angles at the interface with the sample were converted to real and imaginary parts of a dielectric constant by an interpolation method based on dielectric measurements of electrolyte solutions that have conductance near those of investigated samples.19 Dielectric loss values were obtained by subtraction of ionic conductance contribution according to the known formula

′′ ) ′′T - σo/(oω) where ′′, ′′T, σo, o, and ω are dielectric loss, observed imaginary part of complex dielectric constant, ionic conductance of solution, dielectric constant in a vacuum, and angular frequency, respectively. Conductance σo was measured as described in the previous paragraph. A sample holder was thermostated within 0.1 °C. Measurements of permittivity and dielectric loss show a reproducibility of about (1% and (2%, respectively.

Results and Discussion TMAGDC Crystal Structure. Crystal data: N(CH3)4+ + C26H42NO5- + 5H2O, orthorhombic, P212121, a ) 11.436(2) Å, b ) 38.624(5) Å, c ) 8.012(1) Å, V ) 3539(1) Å3, Z ) 4, Do ) 1.14 g cm-3, Dc ) 1.15 g cm-3, µ ) 0.7 mm-1, mp ) 438-439 K. Thermogravimetric analysis gives 4.8 H2O in the asymmetric unit. All non-H atoms were located by Fourier syntheses. TMA+ carbon atoms and an oxygen atom of a water molecule showed rather high thermal parameters and atomic coordinate estimate standard deviations (esd’s) that reflect a situation due to the low number of observed reflections. (19) Athey, T. W.; Stuchly, M. A.; Stuchly, S. S. IEEE 1982, MTT 30, 82.

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Table 1. Side Chain and Ring D Torsion Angles (deg) Together with ∆ and Om (deg)a of TMAGDC,b NaGDC, CaGDC (Two Anions),c NaGC, and RbGC C13-C17-C20-C22 C13-C17-C20-C21 C17-C20-C22-C23 C20-C22-C23-C24 C22-C23-C24-O27 C22-C23-C24-N28 C23-C24-N28-C29 O27-C24-N28-C29 C24-N28-C29-C30 N28-C29-C30-O31 N28-C29-C30-O32 C13-C14 C14-C15 C15-C16 C16-C17 C13-C17 ∆ φm

TMAGDC

NaGDC15

CaGDC21

CaGDC21

NaGC7

RbGC22

172 (1) -63 (1) 64 (1) -173 (1) 133 (1) -47 (1) -179 (1) 1 (1) 88 (1) -174 (1) 3 (1) 47 (1) -37 (1) 12 (1) 17 (1) -38 (1) 4 47

-166 -39 -173 -166 53 -123 178 2 -158 -20 168 44 -32 8 19 -38 14 44

175 -58 -161 177 -9 174 177 0 -118 -31 153 45 -32 7 20 -39 16 45

174 -63 -167 -174 48 -132 -179 2 86 13 -169 46 -34 7 20 -40 15 46

174 -65 -168 -178 43 -134 176 0 115 -6 174 46 -34 8 20 -40 15 47

-179 -53 64 179 -18 166 179 2 -79 177 0 48 -38 12 17 -39 5 48

a Torsion angle values agree with Klyne and Prelog convention.23 The phase angle of pseudorotation ∆ and the maximum angle of torsion φm are calculated according to ref 20. b esd values in parentheses. c There are two anions in the CaGDC crystal asymmetric unit.

Table 2. Donor‚‚‚Acceptor Distances (Å) up to 3 Å in the Hydrogen Bonds with esd Values in Parenthesesa O25‚‚‚O4i O27‚‚‚O5ii O31‚‚‚O2v O1‚‚‚O4

2.76 (1) 2.84 (1) 2.74 (1) 2.80 (1)

O26‚‚‚O2 N28‚‚‚O25iii O32‚‚‚O1 O1‚‚‚O3

2.72 (1) 2.87 (1) 2.79 (1) 2.95 (1)

O27‚‚‚O4ii O31‚‚‚O1iv O32‚‚‚O3iv O2‚‚‚O3

2.82 (1) 2.75 (1) 2.72 (1) 2.72 (1)

a Symmetry codes: (i) 1 + x, y, 1 + z; (ii) x, y, 1 + z; (iii) x - 1, y, z - 1; (iv) x - 1/2, 1/2 - y, 1 - z; (v) x - 1, y, z.

Figure 3. TMAGDC crystal packing viewed along c.

GDC- adopts a ring D conformation very close to the half-chair symmetry,20 as in rubidium glycocholate (RbGC) case (Table 1). Its side chain is in a gauche conformation up to C24 and shows an overall conformation different from those of GDC- and glycocholate anions listed in Table 1. A side chain flexibility and ability to change its elongation is once more observed, thus confirming its important role in the formation of various structural patterns which can suitably interact with several cations. Crystal packing is characterized by a wedge-shaped apolar bilayer, which extends into a plane parallel to ac, containing TMA+ and water molecules within its polar interior (Figure 3). An almost similar bilayer was observed in a calcium deoxycholate crystal structure.21 Alternatively, a polar bilayer can be identified which extends into a plane parallel to ac and is formed by two monolayers (20) Altona, C.; Geise, H. J.; Romers, C. Tetrahedron 1968, 24, 13. (21) D′Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Mazza, F. Langmuir 1997, 13, 3090.

interacting by means of apolar forces. TMA+ and water molecules fill polar channels developed along c and hold together the polar bilayers mainly by means of polar forces. A hydrogen bond network stabilizes crystal packing. Donor and acceptor atoms are listed in Table 2. TMA+ is located in a channel with an inner surface covered by carboxylate, hydroxyl, and carbonyl groups and gives rise with them chiefly to polar interactions (especially ion-ion and ion-dipole). The strongest ones, ranging from 3.2 to 3.6 Å, are shown in Figure 4 together with those of Na+ observed in a NaGDC crystal structure.15 A comparison between coordinations of the two ions points out that Na+ potential energy is lower than that of TMA+ because TMA+ charge is distributed on a greater volume and five Na+‚ ‚ ‚oxygen distances are shorter than the carbon‚ ‚ ‚oxygen ones. Thus, Na+ is more firmly anchored to GDC- aggregates than TMA+. CD Spectra of BR with NaGDC and TMAGDC. Many years ago it was observed that BR becomes optically active on binding to NaDC in aqueous solution.24 Later, the induced optical activity of BR was explained by invoking the helical structure proposed for NaDC micellar aggregates.25 Earlier studies showed the validity of this hypothesis verified also for other bile acid salts.6,8,15,22,26-29 Helices and other chiral objects interact preferentially (22) D’Archivio, A. A.; Galantini, L.; Gavuzzo, E.; Giglio, E.; Scaramuzza L. Langmuir 1996, 12, 4660. (23) Klyne, W.; Prelog, V. Experientia 1960, 16, 521. (24) Perrin, J. H.; Wilsey, M.J. Chem. Soc., Chem. Commun. 1971, 769. (25) Reisinger, M.; Lightner, D. A. J. Inclusion Phenom. 1985, 3, 479. (26) D’Alagni, M.; Delfini, M.; Galantini, L.; Giglio, E. J. Phys. Chem. 1992, 96,10520. (27) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (28) D’Alagni, M.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 5811. (29) Bonincontro, A.; Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. J. Phys. Chem. B 1997, 101, 10303.

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Figure 4. TMA+ and Na+ coordination in TMAGDC and NaGDC crystal structures. The carbon‚ ‚ ‚oxygen and Na+‚ ‚ ‚ oxygen distances (Å) up to 3.6 Å are shown.

with one BR enantiomeric conformer, giving rise to a bisignate CD Cotton effect. The above mentioned studies showed that CD spectra of dihydroxy salts differ remarkably from those of trihydroxy ones and indicated the formation of dissimilar interaction complexes between BR and dihydroxy or trihydroxy salt micellar aggregates, in agreement with their different structures. Therefore, BR chiral recognition is sensitive to micellar aggregate structures. CD spectra of BR in aqueous micellar solutions of dihydroxy salts generally showed a preferential complexation of left-handed BR enantiomer as inferred by a (+) shorter wavelength Cotton effect followed by a (-) longer wavelength Cotton effect on the basis of the exciton coupling theory.30 BR enantioselective complexation to NaGDC and TMAGDC has been investigated in aqueous solutions to check the similarity between their micellar aggregate structures. Some typical CD spectra recorded as a function of pH are reported in Figure 5. They present two dichroic bands of opposite sign centered at about 410 and 465 nm and a crossover point at about 435 nm. A slight preferential complexation of left-handed BR enantiomer is suggested by a weak bisignate CD Cotton effect. The low BR concentration (4.6 × 10-6 M) ensures that BR is present (30) Blauer, G.; Wagnie´re, G. J. Am. Chem. Soc. 1975, 97, 1949.

D’Archivio et al.

Figure 5. CD spectra of BR (4.6 × 10-6 M) with 0.05 M NaGDC (top) or TMAGDC (bottom) in aqueous solutions at pH 7.3 (a), 7.6 (b), 8.1 (c), 8.4 (d), and 8.9 (e).

mainly as monomer, owing to its well-known tendency to associate upon increasing concentration and as a function of pH.31-33 On the other hand, pH increase causes BR conformational changes passing from biacid species to monoanionic and dianionic ones. This phenomenon is supported by the progressive flattening of CD spectra with the pH increasing. Changes of their profiles at higher pH values, where BR dianions predominate, can be attributed to an intramolecular coupling between electric transition dipole moments of the two pyrromethenone chromophores in the dianion different from that in the biacid and/or to an intermolecular dipole-dipole coupling between dianions which could be partially protonated. A new band appears at about 490 nm which was observed in concentrated aqueous alkaline solutions of BR and attributed to a self-association process.31,33,34 BR-NaGDC and BR-TMAGDC CD spectra are very similar. They also show a very close resemblance with (31) Brodersen, R. Acta Chem. Scand. 1966, 20, 2895. (32) Hansen, P. E.; Thiessen, H.; Brodersen, R. Acta Chem. Scand., Part B 1979, 33, 281. (33) Carey, M. C.; Koretsky, A. P. Biochem. J. 1979, 179, 675. (34) Brodersen, R. CRC Crit. Rev. Clin. Lab. Sci. 1979, 11, 305.

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Figure 6. Rh of 0.10 M NaGDC or TMAGDC aqueous solution as a fuction of NaCl or TMACl concentration. Average standard deviation is (0.3 Å.

Figure 7. Rh of aqueous solutions containing 100 mM of GDCand 600 mM of Na+ + TMA+ as a function of Na+ concentration. Average standard deviation is (0.3 Å.

those of NaDC,26 NaTDC,27 RbTDC,27 and CaTDC.6 Because BR interacts with the outer lateral surface of NaDC (8/1 helix), NaTDC, RbTDC, CaTDC, and NaGDC (7/1 helices) anion helical structures, anion structures of TMAGDC and NaGDC micellar aggregates should be very similar. emf measurements on NaGDC aqueous solutions containing TMACl12 gave further information. Trimer persistent presence, even when oligomers of greater aggregation number disappear by increasing ionic strength as well as micellar size, was strong evidence in favor of the 7/1 helix formed by trimers. Although there was a swamping excess of TMA+, Na+ seemed to have a greater affinity than TMA+ for GDC- aggregates, since Na+ fractional binding is higher than 0.5.12 As a consequence, the free energy gain associated with Na+ transfer from bulk solution to micellar aggregate is greater than in the case of TMA+. Similar conclusions were reached for NaTDC.5 QELS Study of TMAGDC and NaGDC. In Figure 6 Rh of 0.10 M NaGDC or TMAGDC aqueous solution is reported versus NaCl or TMACl concentration. NaGDC and TMAGDC Rh values are practically equal up to a 0.2 M NaCl or TMACl concentration. NaGDC values show a much faster increase than those of TMAGDC at higher NaCl concentration. This finding suggests that Na+ has a greater affinity than TMA+ for GDC- aggregates and agrees with previous results, which indicated a decrease of cation affinity for anion aggregates by decreasing cation charge density. For instance, Rh values showed the following trend versus ionic strength: NaGDC > RbGDC > TMAGDC; NaTDC > RbTDC > tetramethylammonium taurodeoxycholate;5 LiDC > NaDC > KDC > RbDC.4 In the case of deoxycholic acid salts, the X-ray analysis of their fibers supported this affinity order.4 Moreover, inspection of Figure 6 points out that NaGDC + TMACl and TMAGDC + TMACl give rise to nearly equal Rh values, although Na+ fractional binding in similar conditions is higher than 0.512 and Na+ > TMA+ affinity for GDC- aggregates. Thus, TMA+ binding to GDCaggregates could give rise to formation of TMAGDC aggregates smaller than NaGDC ones and could interrupt NaGDC aggregate growth. Other QELS measurements have been performed to get information on Na+ and TMA+ relative affinity for GDC- aggregates. Aqueous solutions containing GDC(100 mM) and NaCl + TMACl in such a way as to keep constant the sum of Na+ + TMA+ concentrations (600 mM) have been investigated. The plot of Rh against Na+ concentration is depicted in Figure 7. The experimental points should lie on the straight line passing for those

corresponding to Na+ concentration 0 and 600 mM (Figure 7) assuming nearly equal free energy gains associated with Na+ and TMA+ transfer only (the anion being the same for NaGDC and TMAGDC) from the bulk solution to the micellar aggregates, as it was observed for some deoxycholic acid salts.4 In this case the Na+ or TMA+ populations in the micellar aggregates are linearly proportional to their molar fractions. However, because the experimental points lie below the straight line (Figure 7) the observed trend indicates that TMA+ influences Rh values more than Na+. Moreover, also a small TMA+ concentration causes a fairly remarkable decrease of Rh values as shown by the data within the Na+ concentration range 450-600 mM. These findings support the hypothesis that TMA+ binding to NaGDC aggregates interrupts their growth. Electrolytic Conductance Study of TMAGDC. Models of trihydroxy or dihydroxy salt micellar aggregates provide both anion structures and cation locations9,10 or only anion structures,3-5,27 respectively. Cation-anion interactions in trihydroxy salts are very strong, whereas interactions in dihydroxy salts (7/1 and 8/1 helices) are unknown. Previously, electrolytic conductance measurements were performed on NaDC,4 NaTDC,4,10 NaGDC,10 NaTC,10 and NaGC10 aqueous solutions, containing NaCl at concentrations ranging from 0 to 0.8 M, to throw light on Na+ binding to anion aggregates, since conductance is affected by ion interaction strength. Specific conductance practically did not depend on the particular salt, so that dihydroxy and trihydroxy salts behaved similarly. Bile salt contribution to conductance decreased by increasing NaCl concentration, was approximately zero around NaCl concentration 0.5-0.6 M, and became negative at higher concentrations. Results suggested that Na+ strongly interacts with anion aggregates, reinforces its interactions when micellar size increases, and together with Cl-, coming from NaCl, could be included into micellar aggregates, especially at high ionic strength. Moreover, obstruction effect due to micellar size and shape was discarded because dihydroxy and trihydroxy salts have nearly the same specific conductance values and very different micellar sizes and shapes, mainly at high ionic strength.10 Here TMAGDC has been investigated by adding TMACl from 0 up to 0.8 M concentration and compared with NaGDC (Figure 8). Conductance trends of TMAGDC + TMACl and NaGDC + NaCl as compared with those of TMACl and NaCl, respectively, are very similar. Thus, the above-mentioned conclusions reported for sodium salts10 are valid for TMAGDC. Surprisingly, TMAGDC + TMACl conductance is greater than NaGDC + NaCl conductance up to ∼0.2 M TMACl

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Figure 8. Specific conductance of NaGDC + NaCl or TMAGDC + TMACl as a function of added NaCl or TMACl, respectively. NaGDC and TMAGDC concentration is 0.10 M. Those of NaCl and TMACl are fitted by straight lines (their regression values are both 0.99) and are shown for comparison.

concentration, where it equals that of NaGDC + NaCl, and subsequently is progressively lower. Because ionic mobility of TMA+ < Na+ in water, a greater TMAGDC + TMACl conductance at low ionic strength can be explained assuming that TMA+ interacts more weakly than Na+ with GDC- aggregates. At higher ionic strength NaCl and TMACl contributions to conductance predominate over those of NaGDC and TMAGDC, so that conductance difference between NaGDC + NaCl and TMAGDC + TMACl is approximately equal to that between NaCl and TMACl (Figure 8). Furthermore, methyl group apolar character suggests that TMA+ can be bound by means of hydrophobic forces with the apolar lateral surface of GDCaggregates, covered by C18 and C19 methyl groups (Figure 2), thus decreasing even more the conductance. Dielectric Study of TMAGDC. Complex dielectric constant * ) ′ - j′′ has been obtained as a function of frequency at 15, 25, 35, and 45 °C on TMAGDC aqueous solutions within the concentration range 0.01-0.10 M. As an example, permittivity ′ and dielectric loss ′′ of 0.06 M samples are shown as a function of frequency at 15, 25, 35, and 45 °C in Figure 9. Experimental point trends are characteristic of dielectric relaxation, usually termed β dispersion, which is observed in the frequency range 106-109 Hz in macromolecular solutions.35-38 Best fitting lines are shown (Figure 9) in terms of a Cole-Cole dispersion applying the known equation

* ) ∞ + ∆/[1 + (jωτ)1-R] (35) Grant, E. H.; Keefe, S. E.; Takashima, S. J. Phys. Chem. 1968, 72, 4373. (36) Takashima, S.; Gabriel, C.; Sheppard, R. J.; Grant, E. H. Biophys. J. 1984, 46, 29. (37) Bonincontro, A.; Caneva, R.; Pedone,F. J. Non-Cryst. Solids 1991, 133, 1186. (38) Pedone, F.; Bonincontro, A. Biochim. Biophys. Acta 1991, 1073, 580.

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Figure 9. Permittivity ′ (top) and dielectric loss ′′ (bottom) of 0.06 M TMAGDC aqueous solution versus frequency at 15, 25, 35, and 45 °C.

where ∞, ∆, τ, and R are high-frequency limit permittivity, dielectric increment, relaxation time, and an empirical parameter that indicates a spread of relaxation times, respectively. Disagreement in the frequency region near 1 GHz (Figure 9, bottom) originates from water contribution to dielectric loss, especially for lower temperatures. Apparent electric dipole moment of solute is evaluated from the dielectric increment by means of the Oncley formula,39,40 verified for solutes of molecular weights comparable with those of bile salt micellar aggregates41-44

µ2 ) 2kToM ∆/Noc where µ, k, T, M, No, and c are “effective” electric dipole moment, Boltzmann constant, absolute temperature, molecular weight of a bile salt monomer, Avogadro’s number, and solute concentration in g m-3, respectively. The Oncley formula assumes that dielectric relaxation is caused by orientational polarizability of polar molecules or molecular assemblies in solution. For instance, cations, anion aggregates, and their coordinated water molecules can move and can be oriented when an electric field is applied. The “effective” µ value is calculated in the limit of noninteracting micellar aggregates for bile salt monomer in its specific aggregation distribution function and not for micellar aggregates, M being the monomer molecular weight. Therefore, µ is the average electric dipole moment per monomer. It must be noticed that dielectric results (39) Oncley, J. L. In Proteins, Amino Acids and Peptides; Cohn, E. J., Edsall, J. T., Eds.; Reinhold: New York, 1943; p 543. (40) Pethig R.; Kell, D. B. Phys. Med. Biol. 1987,32, 933. (41) Takashima, S. In Physical Principles and Techniques of Protein Chemistry, Part A; Leach, A. J., Ed.; Academic: New York, 1969; p 291. (42) Gerber, B. R.; Routledge, L. M.; Takashima, S. J. Mol. Biol. 1972, 71, 317. (43) Grant, E. H.; South, G. P.; Walker,I. O. Biochem. J. 1971, 122, 765. (44) Pethig, R. In Dielectric and Electronic Properties of Biological Materials; Wiley: Chichester, U.K., 1979; pp 70-99.

TMA+ Influence on Micellar Aggregate Formation

Figure 10. Average electric dipole moment per TMAGDC and NaGDC monomer (µ) versus concentration (c, mM) at 15 °C. Standard deviations are shown.

Figure 11. Average electric dipole moment per TMAGDC monomer (µ) versus temperature at concentrations 0.02, 0.06, and 0.10 M.

refer to solutions without TMACl, containing small size aggregates (Figure 6). Values of µ at 15 °C only are reported versus TMAGDC concentration in Figure 10, because they can be considered independent of temperature at a fixed concentration within the range 15-45 °C, bearing in mind their standard deviations. As an example, µ values of TMAGDC samples at concentrations 0.02, 0.06, and 0.10 M are shown as a function of temperature in Figure 11. Probably, each sample has the same, or nearly the same, composition, population, and structure of micellar aggregates within the range 15-45 °C. NaGDC µ values10 are the closest to TMAGDC ones among those available up to now (NaTDC,10,29 NaGDC, NaTC, NaGC,10 and NaDC4) and are depicted for comparison in Figure 10, where the NaGDC 0.01 M datum is not reported because of its unfavorable signal-to-noise ratio. TMAGDC shows high µ values, greater than those of NaGDC. The TMA+ 10-fold coordination to oxygen atoms (five belonging to water molecules), observed in the above-mentioned TMAGDC crystal

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structure (Figure 4), supports that the high µ values could be accounted for by a contribution of cation hydration water besides that of the anions. High µ values agree with our model10 and permit the discarding of aggregate structures that are disordered or have a center or a pseudocenter of symmetry, because they correspond to low or zero µ values. Generally, µ values decrease passing from monomeric species to bigger aggregates on increasing TMAGDC concentration (Figure 10) in agreement with monomer decreasing and ordered aggregate (for instance, helices) growth. Very probably the electric dipole moment of a single monomer is very high because is not lower than that of the 0.01 M TMAGDC sample (about 70 D), where, although very likely monomers prevail, other species with lower µ can be present. Therefore, each TMA+ is anchored to one anion in dilute solution, giving rise to ion pair formation. A similar behavior is displayed by NaTDC,10,29 NaGDC, NaTC, NaGC,10 and NaDC.4 TMAGDC experimental points of Figure 10 show a maximum at 0.06 M, at variance with those of NaGDC. Similar situations were observed for NaDC,4 NaTC, and NaGC10 and were interpreted assuming a two-structure model and equilibrium. First and second structures are polydisperse systems which predominate in a region of the concentration ranges 0-0.05 M and 0.05-0.10 M, respectively. The increase of µ values in the range 0.050.06 M can be explained by assuming that in this region a second structure begins to form and its µ is greater than that of the first structure. It must be stressed, however, that in the absence of the maximum the same explanation can be invoked, except that the second structure µ is not greater that the first structure one. An indication of first and second structure compositions is provided by the following: TMAGDC relaxation frequencies are greater than those of NaGDC, according to the smaller size of TMAGDC aggregates. emf measurements of NaGDC + TMACl aqueous solutions as a function of pH (7-10), ionic strength, and bile salt concentration.12 Na3(GDC)3 trimers and a very small amount of NaH(GDC)2 dimers are always present in the pH range 7-10 and 7-8, respectively. Because TMAGDC samples have pH ≈ 7 and TMAGDC micellar size is smaller than that of NaGDC at a fixed concentration, it is highly probable that the number of TMAH(GDC)2 is greater than that of NaH(GDC)2 at the lowest concentrations. Therefore, an equilibrium between dimers (first structure main component) on one hand and trimers together with aggregates formed by trimers (second structure main components) on the other can be invoked. As far as NaGDC is concerned, its experimental points can be fitted by a curve with slope change at a concentration of about 0.05 M (Figure 10), in accordance with a two-structure model and equilibrium proposed for NaTDC.10,29 First and second structure are polydisperse systems mainly characterized by oblate aggregates (from one to four trimers) and cylindrical aggregates (from five trimers upward, 7/1 helix), respectively. In conclusion, X-ray, CD, QELS, electrolytic conductance, and dielectric results converge in outlining similar structural models (7/1 helices) for bigger TMAGDC and NaGDC anion aggregates, although TMAGDC micellar size is smaller than that of NaGDC at a fixed concentration and ionic strength. Both cations strongly interact mainly by means of polar forces with anion aggregates, Na+ being more firmly bound to GDC- aggregates than TMA+. TMA+ addition to NaGDC probably interrupts its aggregate

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growth. Therefore, emf data can be used to get information on NaGDC micellar aggregate structure. Very probably, the same conclusion is valid for NaTDC, NaDC, and NaTC, for which emf data are available.11,13,14 Acknowledgment. This work was sponsored by Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica. Professor A. Bonincontro is gratefully acknowledged for helpful discussions.

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Supporting Information Available: Listings of observed and calculated structure factors, atomic fractional coordinates together with equivalent isotropic thermal parameters (Table 1), bond lengths and bond angles (Table 2), and relaxation frequency fo, dielectric increment ∆, and average electric dipole moment per monomer µ for TMAGDC aqueous solutions at 15 °C (Table 3). This material is available free of charge via the Internet at http://pubs.acs.org. LA001704P