J. CHEM. SOC. FARADAY TRANS., 1994, 90(11), 1523-1532 Micellar Aggregates of Sodium Glycocholate and Sodium Taurocholate and their Interaction Complexes with Bilirubin-IXa Structural Models and Crystal Structure Maria D'Aiagni" Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive del C.N.R., c/o lstituto di Chimica, Universita Cattolica , Largo F. Vito I,00 168 Roma , Italy Lucian0 Galantini and Edoardo Giglio" Dipartimento di Chimica, Universita di Roma 'La Sapienza ', P.le A. Moro 5,00185Roma, Italy Enrico Gavuzzo lstituto di Strutturistica Chimica 'G. Giacomello ' C.N.R., C.P. no. 10,00016 Monterotondo Stazione, Roma, Italy Lucio Scaramuzza lstituto di Teoria e Struttura Elettronica e Comportamento Spettrochimico dei Composti di Coordinazione del C.N.R., C.P.no. 10,00016Monterotondo Stazione, Roma, Italy Sodium glycocholate (NaGC) and taurocholate (NaTC) have been studied by means of X-ray and circular dichro- ism (CD) measurements, using bilirubin-lXa (BR) as probe molecule, together with potential-energy calculations. Helical models for the micellar aggregates of NaGC and NaTC were inferred from crystal structures solved by X-ray analysis. Since it is known that chiral molecules, micellar aggregates and macromolecules select prefer- entially or exclusively one of the two enantiomeric conformers of BR, CD spectra of BR in submicellar and micellar aqueous solutions of NaGC and NaTC were recorded as a function of pH and BR concentration in order to verify these helical models and the enantioselective ability of the bile salt monomers and micellar aggre- gates.Potential-energy calculations supported the CD experimental results and provided reasonable bile salt-BR interaction models. The behaviour of NaGC and NaTC is compared with that of sodium deoxycholate (NaDC), previously studied. The CD spectra of the bile salt-BR systems seem to allow characterisation of the typical structure of the bile salt micellar aggregates. Helical structures have been proposed for NaDC and rubid- ium deoxycholate (RbDC), and proved by means of wide-and small-angle X-ray scattering, '-'EXAFS,4*5 NMR,2,6-8 EPR' and CD8-" measurements together with potential- energy calculation^.^^^*^^^^ Subsequently, some studies on sodium glycodeoxycholate (NaGDC),' 'sodium taurodeoxy- cholate (NaTDC),7*'0-12 NaGC'' and NaTC14 were accom- plished following the strategy used for NaDC and RbDC." Again, helical models were found in crystals and their ability to describe aqueous solutions of micellar aggregates for NaGDC and NaTDC was checked. Although some evidence was collected in favour of these models, no definitive proof about their consistency was reached.We previously used probe molecules to investigate the nature of the interactions between bile salt and probe, and to obtain information on the structure of the micellar aggre- gate~.~*'*~-'~BR, a bile pigment of biological interest,16 gives rise in solution to an equal number of two interconverting enantiomers in dynamic equilibrium having left ( -) and right (+) handed chirality (LBR and RBR, respectively). BR is characterized by a 'ridge tile' conformation stabilized by six intramolecular hydrogen bonds (Fig.l), and has a structure which was determined mainly from X-rayI7-' andNMR20-23 measurements. The enantiomers interconvert by breaking and remaking the six intramolecular hydrogen bonds. The dynamic equilibrium is disrupted by a preferential interaction of BR with a chiral molecule, macromolecule or molecular assembly. In this case, BR exhibits optical activity, giving rise to a bisignate CD Cotton effect, on the basis of the exciton coupling theory,24 typical of dipole-dipole coupling between the two pyrromethenone chromophores of one BR molecule.Many interaction complexes of BR with a wide variety of compounds were investigated by means of CD.25 It was observed that the complexation of NaDC with BR in water produces a bisignate CD Cotton effect,26 which was explained later by invoking a chiral structure of the helical micellar aggregates of NaDC.27 Bisignate CD spectra were also recorded for the complexes of BR with RbDC, NaGDC and NaTDC in water.'' More recently, the influence of pH and BR concentration on the CD spectra of submicellar and micellar NaDC-BR aqueous solutions was investigated.8 Potential-energy calculations provided some NaDC-BR interaction models which were checked by CD and NMR measurements. The aim of this paper is to obtain information on the struc- ture of the NaGC and NaTC micellar aggregates, since NaGC and NaTC (Fig.1) are the most abundant and the most carefully studied conjugated bile salt in humans, respec- tively. We resort to structural models, observed in crystals, in order to represent and to verify the micellar aggregates in aqueous A possible model, composed by bile salt molecules arranged in a helix with 2, symmetry, is avail- able for NaGC.13 NaTC is known to form three crystalline phases.28 We crystallized the two most stable, belonging to the triclinic and monoclinic systems. The crystal structure of the triclinic phase has been solved previou~ly.'~ The struct- ural unit is a bilayer which is unlikely to grow in solution by addition of molecules one at a time.On the other hand, several experiments have suggested that the bile salts self- associate in a non-cooperative continuous manner in water and at low ionic strengths, whereas sometimes a cooperative association of a large number of molecules occurs, especially at high ionic strength^.^' In the case of NaTC, for example, a progressive aggregation can be inferred from reliable experi- mental data obtained by means of surface tension and trans- lational diffusion coefficient measurement^.^^ Thus, a helix C(18) C(21) 4 O(32) C(18) C(21) 0(33) 5% 0(34) O(35) Fig. 1 Atomic numbering and enantiomeric conformers of BR (top). Broken lines represent intramolecular hydrogen bonds. Atomic num- bering of NaGC (middle) and NaTC (bottom).Hydrogen atoms are omitted. fulfils both types of growth, since it is a one-dimensional open system containing monomers with unsaturated forces at the end-points if the solvent molecules are disregarded." The helix can grow by a stepwise addition of monomers or by welding of helices. Therefore, the structural unit found in the NaGC crystal is compatible with the experimental results, whereas that found in the triclinic NaTC crystal is less prob- able. For these reasons we solved the structure of the NaTC monoclinic crystal, hoping to obtain a reliable model for the micellar aggregates, and investigated by means of CD mea- surements and potential-energy calculations the complexes of BR with NaGC and NaTC to commence a study of the tri- hydroxy bile salts.Experimental Materials and Methods NaGC and NaTC (Sigma) were twice crystallized from a mixture of water and acetone. BR (puriss., cu. 99%, Fluka) was used. High-performance liquid chromatography (HPLC) and spectroscopic measurements indicate that the com-mercial sample contains at least 96% of bilirubin-IXa. Trizma base (reagent grade) and phosphate buffer (pure reagent) were purchased from Sigma and Carlo Erba, respec- tively, and were used in order to adjust the pH. BR was J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 added to NaGC and NaTC aqueous solutions by using 0.01 mol dm-3 aqueous NaOH. All the measurements were accomplished after 30 min of preparation. CD spectra were recorded on a JASCO J-500A spectropol- arimeter at 25°C by using quartz cells with pathlengths of 0.1, 0.2,0.5, 1.0 and 2.0 cm and by flushing with dry ultrapuri- fied 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 x mol dm-3 in dioxane) on the basis of a molar ellipticity [OlZo4 11 180 degree cm2 dmol-'. = Potential-energy calculations were carried out on Vax 8530 and MicroVax I1 computers by means of programs written in our laboratory. Suitable single crystals of monoclinic NaTC were grown from a solution containing water, acetone and diethyl ketone by vapour diffusion of acetone into the solution. Intensity X-ray data were collected at room temperature by means of a Syntex P2, automatic four-circle diffractometer, equipped with a graphite monochromator, using Cu-Ka radiation (A = 1.5418 A).Results and Discussion Crystal Structure of Monoclinic NaTC Unit cell parameters were determined by least-squares refine- ment of the angular setting of 15 selected reflections. Crystal data: 2NaC2,H4,N0,S + 11.5H20 + C,H,O, f, = 1340.6, monoclinic, C2, a = 40.204 (lo), b = 7.665 (2), c = 23.042 (7)A, jl = 92.77 (2)", I/ = 7092 (3) A3, 2 = 4, D,= 1.26 g ~m-~, Do= 1.28 g (by flotation in a chloroform-benzene chloride solution), p = 14.31 cm-', mp 499 K. A total of 4977 unique reflections with I > 2.5o(I) were considered observed and used in the calculations. They were collected at a variable and appropriate speed in o scanning mode to a maximum 28 of 138".Background counts were taken for a time equal to that of the scan. The intensities of three standard reflections, monitored every 100 reflections, remained essentially constant throughout data collection. The data were corrected for Lorentz and polarization effects, but not for absorption. The structure was solved by using the MULTAN pr0gram.j' The refinement was carried out by means of the program SIR-CAOS.32 The final atomic coordinates and equivalent isotropic thermal parameters, together with their estimated standard deviations (esd) in parentheses, have been deposited. The atom labelling in accordance with Fig. 1 and 2, where bond lengths and bond angles are given.Atomic numbering from O(1) to O(12) refers to oxygen atoms of water molecu1es.t Scattering factors and anomalous disper- sion coefficients were taken from ref. 33. The hydrogen atoms were generated at the expected positions, except for the water, hydroxylic and acetone hydrogens which were not taken into account in the calculations. A full-matrix least- squares refinement was performed with anisotropic tem-perature factors for all the non-hydrogen atoms, except for C(66), O(7) and the atoms of the acetone molecule, which appeared in the Fourier syntheses with electron densities lower than those of similar atoms, and during the refinement exhibited some abnormally high anisotropic temperature factors. O(7) was refined isotropically. C(66) was refined iso- tropically with fixed atomic coordinates, whereas the acetone atoms were introduced with fixed atomic coordinates and t Final fractional coordinates and equivalent isotropic thermal parameters of the non-hydrogen atoms have been deposited with the Cambridge Crystallographic Data Centre.Details are available from the Editorial Ofice. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 0s 00 oc ON C( 1)-C( 10)-c(9) C(5)-C( 10)-C( 19) C( 12)-C( 13)-C( 17) C(14)-C(13)-C(18) C(31)-S(32)-0(34) O(33)-S(32)-0( 35) C(36)-C(45)-C(44) C(4O)-C(45)-C(54) C(47)-C(48)-C(52) C(49)-C(48)-C(53) C( 66)-S(67)-0(69) 0(68)-S(67)-0(70) Q 111.6 109.8 1 16.4 113.3 107.9 109.4 11 1.4 110.3 118.0 113.5 107.7 11 1.9 Fig.2 Final bond lengths (A)and bond angles (degrees) found for the two taurocholate anions of the asymmetric unit. The average esds are 0.012 A and 0.7"with maximum values of 0.021 A and 1.3". isotropic thermal parameters. An occupancy factor of 0.5 was assigned to O(7) and to the acetone atoms. The location of the acetone atoms is very approximate owing to their occu- pancy factor of 0.5 and to a possible positional disorder, sup- ported by the low electron density of the corresponding peaks observed in the Fourier syntheses. However, the inclu- sion of acetone, located near a special position, was crucial for fitting some strong low-angle reflections and for obtaining a satisfactory convergence in the refinement.The atomic coordinates of the hydrogens were not refined, and their thermal parameters were taken to be equal to the isotropic ones of the parent atoms. The function minimized was w( I F, I -I F, I )2 with w = (sin 6/A)2.Several other weighting schemes were checked, but the results were worse. The final agreement factors R and R, converged to 0.074 and 0.083, respectively. S was found to be 0.68. The torsion angles of the side chain and ring D are re- ported in Table 1. The two NaTC anions are characterized by a conformation of ring D intermediate between the half- chair and the C(13) en~elope.~' The C( 13)-C( 17)-C(20)- C(21), C( 13)-C( 17)-C(20)-C(22), and C( 17)-C(20)-C(22)- C(23) torsion angles are always confined within a narrow range around -60 and 180", respectively, as established by energy calculation^.^^*^^ The deviation from planarity of the amide group (assuming that the three bonds to the nitrogen atom lie in a plane) is slight.The rotation around N(29)-C(30) seems to be almost completely free, since the values of C(24)-N(29)-C(3O)-C(31) vary from about -160 to 120" in all the conjugated bile salts studied so far.'0~1'*13*14From these results it emerges that several permissible conforma- tions can be achieved in order to satisfy packing and energy requirements in the crystals and in solution. The coordination of the sodium ions and oxygen atoms of water molecules is visible in Fig. 3. The sodium ions are hexacoordinated, with a distorted octahedral coordination.They are engaged in strong Coulombic interactions with the SO, -groups and ion-dipole interactions with water mol- ecules. Moreover, the structure is further stabilized by a J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 Table 1 Torsion angles of the NaTC side chain and ring D together with A and 4,,,;esds in parentheses” atoms torsion angleldegrees atoms torsion angle/degrees C(13)-C( 17)4(20)-C(21) C( 13)-C( 17)-c(20)-c(22) C( 17)4(2O)-C(22)-C(23) C(20)-C(22)-C( 23)-C(24) -59.0 (10) 176.6 (6) 178.8 (7) -170.5 (7) C(48)-C(52)-C(5S)-C(56) C(48)-C( 52)-C( 55)-C( 57) C( 52)-C( 55)-C( 57)-C( 58) C(55)-C(57)-C(58)-C(59) -67.7 (10) 166.5 (7) 61.2 (10) -164.6 (8) C( 22)-C(2 3)-C( 24)-O( 28) 43.6 (12) C( 57)-C( 58)-C( 59)-O(63) 66.2 (14) C(22)-C( 23)-C( 24)-N(29) -138.1 (9) C( 57)-C( 58)-C( 59)-N( 64) -116.8 (11) C(23)-C(24)-N(29)4(30) 178.8 (9) C(58)-C( 59)-N( 64)-C( 65) -169.2 (12) C(24)-N(29)-C(3O)-C(3 1) 0(28)-C(24)-N(29)-C(30) N( 29)-C( 30)-C(3 1)-S( 32) C( 30)-C( 3 1 )-S( 32)-O( 33) C(30)-C(3 l)-S(32)-0(34) 94.4 (11) -2.9 (15) 63.3 (10) 51.2 (9) -71.2 (9) C( 59)-N(64)-C( 65)-C( 66) 0(63)-C(59)-N(64)-C(65) N(64)-C(65)-C( 66)-S(67) C( 65)-C(66)-S(67)-0(68) C( 65)-C(66)-S(67)-0(69) -120.1 (13) 7.8 (20) -176.6 (8) 52.3 (8) -73.2 (8) C(30)-C(3 l)-S(32)-0(35) 171.4 (8) C(65)-C( 66)-S(67)-0( 70) 168.6 (8) C( 17)-C( 13)-c( 14)-C( 15) C( 13)-C( 14)-C( 15)-C( 16) C( 14)-C( 1 5)-C( 16)-C( 17) C( 15)-C( 16)-C( 17)-C( 1 3) C( 16)-C( 17)-C( 13)-C( 14) 45.9 (7) -34.1 (8) 8.8 (9) 18.8 (8) -38.8 (7) C( 52)-C(48)-C(49)-C(SO) C(48)-C(49)-C(5O)-C( 51) C(49)-C(5O)-C(5 1)-C( 52) C(50)-C(5 1)-C(52)4(48) C(Sl)-C(52)-C(48)-C(49) 46.3 (8) 3.9 (9) -31.3 (8) 24.1 (9) -42.3 (8) Aldegrees 11.9 24.7 4Jdegrees 46.2 47.4 ” The values of the torsion angles are in accordance with the convention of Klyne and Pre10g.~~ The phase angle of pseudo-rotation, A, and the maximum angle of torsion, 4m,are calculated according to Altona et ~ 1 .~ ~ complex network of hydrogen bonds. Those formed by water molecules are shown in Fig. 3. Two different units can be recognized in the crystal packing (Fig. 4). The first one is a 2, helix, very similar in size and shape to that of NaGC,I3 with an approximate cross-section, perpendicular to the helical axis, which is ellipsoidal if the sodium ions and the water molecules are not taken into account (Fig. 5).The helix presents a polar outer surface around the end regions of the semimajor axis and can be permeable to the aqueous solvent since the separation between two anions related by a b translation along the helical axis is sufficiently large. The most protruding outside apolar groups are the C(18) and C(19) methyl groups. The helix is stabilized by the hydrogen bonds O(25).. aO(35) = 2.741(13), O(26). -vO(28) = 2.785(9), and 0(27)...0(28)= 2.744(13) A, in addition to some of those reported in Fig. 3. Two adjacent anions along the helix are arranged in antiparallel mode and are linked by head-to-tail hydrogen bonds together with a Na(2).. eO(34) Coulombic interaction. The second unit (Fig. 6) resembles the first one in size and shape, although a two-fold rotation axis replaces the two-fold screw axis. The main differences are the side chain conformation (see Table 1) and the location of the hydrated sodium ions, which in the second unit are more embedded between the O(25) hydroxylic group and the sulfonate ion. Two more hydrogen bonds (not shown in Fig. 3) stabilize this unit. They are O(61)-* aO(68) = 2.834(12) and O(62)-. .0(68) = 2.905(13) A. The two facing antiparallel anions are linked by head-to-tail hydrogen bonds, together with a Na(1). -0(70) Coulombic interaction. The crystal packing resembles that of NaGCI3 character- ized by hydrophilic and hydrophobic channels.The hydro- philic channel, centred on a two-fold screw axis at a = 1/4 and c = 1/2, is filled with sodium and sulfonate ions and water molecules, which give rise to strong polar interactions, and is a locus of aggregation of the two types of structural unit. The hydrophobic channel, centred on a two-fold rota- tion axis at a = 0 and c = 0, is filled with acetone molecules, which give rise to van der Waals interactions with the C(18), C(19), C(53)and C(54) methyl groups of NaTC and improve the crystal stability. CD Spectra of NaGC and NaTC with BR It was decided to investigate the enantioselective complex- ation of BR to NaGC and NaTC and to compare the CD results with those of NaDC.8 The spectra of the bile salts, recorded at natural pH within the pH range 6-7, are shown in Fig.7. The trend of Fig. 7(a)and (4,corresponding to a concentration that is certainly below the critical micellar con- centration (c.m.c.), is monotonic and gives rise to molar ellip- ticity values that are always positive or zero up to 250 nm for both NaGC and NaTC. No CD spectrum was observed at higher wavelengths in the region where the absorption of BR occurs. A negative dichroic band appears and becomes deeper upon increasing the concentration, in agreement with the behaviour of NaTDC” and at variance with that of NaDC and sodium ~holate.~~ These findings seem to confirm a non-cooperative continuous self-association of the micellar aggregates, as was observed by means of other techniques for bile salts in water and at low ionic strength.*’ CD spectra at various pHs of NaGC and NaTC below the c.m.c., with BR at a concentration of 4.6 x mol dm-3, are shown in Fig.8. The low BR concentration ensured that BR was present mainly as monomer, because of its well known tendency to associate upon increasing concentration and as a function of pH.39-41 The spectrum of BR with NaGC does not change appreciably within the pH range 7.3- 9.3 (Fig. 8). Its typical profile could indicate a very small pref- erential selection of RBR, as in the case of BR with NaDC.8 Similar results were acquired for BR and NaTC. Only two spectra are shown as examples [Fig. 80 and (g)]. The attempt to investigate the effect of the BR concentration increase as a function of pH in aqueous solutions of NaGC and NaTC was unsuccessful, since BR precipitates at a pH within the range 7-8, beginning from a concentration of ca.lo-’ mol dm-3. CD spectra were recorded only at pH = 9. As an example, the spectrum of BR with NaTC is shown in Fig. 8(h). It is similar to the other ones in this figure and to those recorded for BR with NaDC under nearly the same conditions. Therefore, it seems reasonable to suppose that the NaGC, NaTC and NaDC anions behave likewise in forming the corresponding complexes with BR. This is supported by J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 Fig. 3 Projection on the uc plane of the sodium ions and oxygen atoms of water molecules with their coordination sphere.The distances corresponding to ion-ion, ion-dipole and hydrogen-bonding interactions are given in A,together with their esds in parentheses. the geometry of the NaDC anion-BR interaction complexes, found by means of potential-energy calculations, where the NaDC side chain is weakly engaged with BR (see Fig. 5 of ref. 8). The three bile salt anions are different at the end of the side chain and are practically equal in the remaining part, with the exception that one hydrogen atom of C(7) in NaDC is replaced by one hydroxylic group in NaGC and NaTC. Thus, their complexes with BR could have comparable inter- action energy and those with RBR could be more stable than those with LBR. The behaviour of BR at low concentration (4.6 x loA6mol 0 dm-3) in the presence of NaGC and NaTC micellar aggre- gates is very similar and is shown in Fig.9. The CD spectra are characteristic of a preferential selection of the LBR enan- tiomer at pH values below ca. 7.9. Again, the trend follows that of BR with NaDC, even though in this case the dichroic bands invert at more alkaline pH (about 9, see Fig. 7 of ref. 8). The CD spectra progressively change upon increasing pH, showing that the enantioselective ability of the NaGC and NaTC micellar aggregates is greater with the BR biacid than with the BR dianion and, also, is greater than that of the monomers at pH < 8 (cf: Fig. 8 and 9). The change in the profile of the spectra could be due to an intramolecular coup- ling between the electric transition dipole moments of the two pyrromethenone chromophores in the BR dianion different ow from that in the BR biacid and/or to an intermolecular Fig.4 NaTC crystal packing viewed along b dipole-dipole coupling between BR dianions. The CD 0s 00 Fig. 5 NaTC 2, helix projected (a) along a direction perpendicular to the helical axis, and (b) along the helical axis. A thicker line rep- resents an anion nearer to the observer. Thin full lines indicate ion-ion and ion-dipole interactions. Broken lines indicate hydrogen bonds. spectra of Fig. 10A, compared with those of Fig. 9, show that in the case of NaGC the increase of the BR concentration (3.40xlo-' mol dm-3) is roughly proportional to that of the ellipticity values in the pH range 7.2-9.4.A weak positive 0s Fig. 6 NaTC unit with a two-fold rotation axis projected (a) along a direction perpendicular to the two-fold rotation axis, and (b) along the two-fold rotation axis. The meaning of the lines is as in Fig. 5. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 A/nm Fig. 7 CD spectra of aqueous solutions of NaGC (full line), (a) 1.01 x mol dm-3, pH 6.06;(b) 1.011 x mol dm-j, pH 6.70;(c) 5.059 x mol dmF3, pH 6.98;and NaTC (broken line), (d) 1.02 xlo-' mol dm-3, pH 6.52;(e) 2.500 x mol dm-3, pH 6.33;cf) 5.001 x mol dm-3, pH 6.10.(a) refers to molar ellip- ticity [8] x1O-j. 0.5-I I I I I I 0.25-ca ? 0.0--0.25 -360 380 400 420 440 460 480 500 520 A/nm Fig. 8 CD spectra of BR (4.6xlov6 rnol dm-3) with NaGC (1.00x mol dm-'), (a)pH 7.30;(b) pH 8.10;(c) pH 8.35;(d) pH 9.02;(e) pH 9.32;and with NaTC (1.00x10-3 rnol dmW3), cf) pH7.41;(9)pH 9.06.(h) refers to BR (1.61xlow5mol dm-3) and NaTC (1 .OO x 10-mol dm -3), pH 9.07.J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 Fig. 9 CD spectra of BR (4.6 x loe6 mol dmP3) and NaGC (full line) with molar concentration, (a) 5.012 x lo-’, pH 7.28; (b) 5.010 x lo-’, pH 7.64; (c) 5.002 x lo-’, pH 8.30; (d) 5.005 x pH 8.60; (e) 4.999 x lo-’, pH 9.53; and NaTC (broken line) with molar concentration, cf) 5.007 x pH 7.35; (9)4.997 x lo-’, pH 8.13; (h)4.995 x lo-’, pH 9.12 band centred at 490 nm appears at pH x 7.7 [Fig. 10A(b)]. This band becomes more pronounced and is shifted to the blue upon increasing the pH.Previously, the increase in absorption at about 500 nm was observed in concentrated aqueous alkaline solutions of BR and attributed to an aggre- gation proce~s,~~*~~ which causes a weak intermolecular elec- tric dipole coupling. A similar behaviour is noted for NaTC in Fig. 10A, where only three spectra are shown for compari- son in the same interval of pH. A fourth spectrum at pH 11.60 [Fig. 10AG)] suggests that little change occurs in the bile salt-BR system above pH 9. The effect of a further increase of BR concentration (3.182 x mol dm-3) is dis- played only for NaGC in Fig. 10B, since NaTC precipitates. No sensible influence of BR concentration is observed on the pH value at which the dichroic bands invert, at variance with the NaDC-BR system.’ Poteotiai-energy Calculations The NaGC micellar model was inferred from the tetragonal NaGC crystalI3 and was used to calculate the potential energy of the interaction complex with LBR and RBR in the biacid form, and to check indirectly the validity of the model, without taking into account the water molecules of the solvent.Six NaGC molecules were considered in a right-handed rectangular framework OXYZ and were kept fixed. The atomic coordinates of the first molecule (xl,y,, zl)were obtained from the x, y, z (in A) of Table 1 of ref. 13 as follows: x1 = x -u/4, y1 = y -b/4, z1 = z. Those of the second molecule (x,, y,, z,) are: x, = -xl, y, = -yl, z2 = z1 -42. The other four molecules are generated by trans- posing c for -c in this pair. The hydrogen atoms were put at the expected positions (C-H = 1.08 A, N-H = 1.00 A), except those of the hydroxylic groups, which are inside the helix and interact very weakly with BR, and those of the methyl groups, treated as one atom.The atomic coordinates of BR, in the ‘ridge tile’ conforma- tion of the biacid form,” are reported in Table I1 of ref. 8 for the LBR enantiomer (those of RBR are obtained by changing z for -2). BR was moved as a rigid body by anticlockwise rotations around OX ($1), OY ($,) and OZ (t/j3), in this order, and by translations along OX (tx),OY (t,) and OZ (tz). B I I I I I I I -10 iI -’ I I 1 I , (g), I 360 380 400 420 440 460 480 500 A/nm Fig.10 A, CD spectra of BR (3.40 x mol dm-’) and NLC (full line) with molar concentration, (a) 4.999 x pH 7.25; (b) 5.032 x lov2, pH 7.73; (c) 5.000 x lo-’, pH 7.92; (6)5.013 x lo-’, pH 8.31; (e)5.004 x pH 8.95; (f) 4.996 x lo-’, pH 9.42; and NaTC (broken line) with molar concentration, (9)5.001 x pH7.23; (h) 4.989 x pH 8.15; (i) 5.008 x pH 9.35; 0’)5.009 x lod2,pH 11.60. B, CD spectra of BR (3.182 x mol dm-3) and NaGC with molar concentration, (a) 4.980 x lod2,pH 7.64; (b)5.028 x pH 8.71; (c)4.990 x pH 11.55. The van der Waals energy was computed by using stan- dard atom-atom semiempirical potentials with coefficients listed in Table I11 of ref. 8, together with an N-N potential.42 The hydrogen-bonding energy was calculated by using a direction-dependent 6-4 function43 with parameters equal to those employed in ref.8 and 38. The six-dimensional para- metric space was explored assuming a cut-off distance of 9 A and giving angular and translational increments progressively decreasing from 10 to 2” and from 0.5 to 0.1 A. The two lowest-energy minima for each BR enantiomer are defined in Table 2. For these minima no contribution of the hydrogen- bonding energy was found. Projections perpendicular (along OY) and parallel (along OZ) to the NaGC helical axis of the interaction complexes with BR (minima A-D of Table 2) are depicted in Fig. 11-14, where only four of the six NaGC mol- ecules used in the calculations are shown. The complexes are Table 2 Most relevant data of the lowest-energy minima calculated for the complexes between the NaGC 2, helix and LBR or RBR" minimum ~~ $2 t,b3 tx t,, t, energy A (LBR) 100 0 -43 -11.70 5.65 1.55 -25.82 B (RBR) C(LBR) D(RBR) 265 160 25 2 15 -22 -45 -40 -53 -11.60 -11.40 -10.10 5.90 6.40 7.40 2.30 2.50 0.75 -25.14 -24.98 -24.10 a The rotation angles, translations and energy are given in degrees, A and kcal mol- ',respectively.rather similar and are characterized by stable apolar inter- actions between BR and the B faces of the bile salt anions. The least-squares planes of the two pyrromethenone moieties of BR form a dihedral angle of about lOO", a value nearly equal to that of the dihedral angle formed by the least- squares plane of ring A with that of rings B, C and D in the steroid skeleton.Therefore, a good fit between BR and the rings A, B, C and D can be achieved. The best interactions arise from apolar contacts involving mainly the methyl groups C(18) and C(19) and the most protruding hydrogen atoms of the NaGC helix, together with the methyl and vinyl groups of BR, which are particularly active. The NaGC-LBR complex is more stable than the NaGC-RBR one (compare the energy of minimum A with B and C with D). Thus, a chiral recognition should occur, at least within the range of the pH values characteristic of the BR biacid form, in accord- ance with the CD spectra [(a) and (b)]of Fig. 9 and 10A, and Fig. 10B(a). The small energy difference between the NaGC- LBR and NaGC-RBR complexes suggests a slight prefer- ow 0 Td-.-.__.Fig. 11 Projection perpendicular (a) and parallel (b) to the NaGC helical axis for minimum A of Table 3. A thicker line represents a molecule nearer to the observer. Broken lines indicate hydrogen bonds. J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 r-94 f---..l Fig. 12 As in Fig. 11 for minimum B /-KO PY Fig. 13 As in Fig. 11 for minimum C J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 I e Secondaria, and by the Italian Minister0 per 1'Universita e per la Ricerca Scientifica e Tecnologica. References 1 A. R. Campanelli, S. Candeloro De Sanctis, E. Giglio and S. Petriconi, Acta Crystallogr., Sect. C, 1984,40, 631. 2 G. Conte, R. Di Blasi, E.Giglio, A. Parretta and N. V. Pavel, J. Phys. Chem., 1984,88,5720. 3 G. Esposito, E. Giglio, N. V. Pavel and A. Zanobi, J. Phys. Chem., 1987,91,356. 4 E. Giglio, S. Loreti and N. V. Pavel, J. Phys. Chem., 1988, 92, 2858. 5 E. Burattini, P. D'Angelo, E. Giglio and N. V. Pavel, J. Phys. Chem., 1991,95,7880. 6 G. Esposito, A. Zanobi, E. Giglio, N. V. Pavel and I. D. Camp-bell, J.Phys. Chem., 1987, 91, 83. 7 E. Chiessi, M. D'Alagni, G. Esposito and E. Giglio, J. Inclusion Phenom. Mol. Recognit. Chem., 1991,10,453. Na' '+ Fig. 14 As in Fig. 11 for minimum D ential selection of the LBR enantiomer, in accordance with the low ellipticity values of the CD spectra. 8 M. D'Alagni, M. Delfini, L. Galantini and E. Giglio, J. Phys. Chem., 1992,%, 10520.9 M. D'Alagni, M. L. Forcellese and E. Giglio, Colloid Polym. Sci., 1985,263,160. 10 A. R. Campanelli, S. Candeloro De Sanctis, E. Chiessi, M. D'Alagni, E. Giglio and L. Scaramuzza, J. Phys. Chem., 1989,93, 1536. 11 A. R. Campanelli, S. Candeloro De Sanctis, E. Giglio and L. Scaramuzza, J. Lipid Res., 1987,243,483. 12 M. D'Alagni, E. Giglio and S. Petriconi, Colloid Polym. Sci., 1987, 265, 517. 13 A. R. Campanelli, S. Candeloro De Sanctis, L. Galantini and E. Giglio, J. Inclusion Phenom. Mol. Recognit. Chem., 1991,10,367. 14 A. R. Campanelli, S. Candeloro De Sanctis, A. A. D'Archivio, E. Giglio and L. Scaramuzza, J. Inclusion Phenom. Mol. Recognit. Chem., 1991,11,247. 15 A. R. Campanelli, S. Candeloro De Sanctis, E. Giglio, N.V. Pavel and C. Quagliata, J. Inclusion Phenom. Mol. Recognit. Chem., 1989,7,391. 16 Bile Pigments and Jaundice: Molecular, Metabolic and Medical Aspects, ed. J. D. Ostrow, Marcel Dekker, New York, 1986. 17 R. Bonnett, J. E. Davies, M. B. Hursthouse and G. M. Sheldrick, Proc. R. SOC.London, Ser. B, 1978,202,249. 18 G. Le Bas, A. Allegret, Y. Mauguen, C. De Rango and M. Bailly, Acta Crystallogr., Sect. B, 1980,36, 3007. 19 A. Mugnoli, P. Manitto and D. Monti, Acta Crystallogr., Sect. C, 1983,39, 1287. 20 D. Kaplan and G. Navon, Biochem. J., 1982,201,605.The energies of the A-D minima, computed with a cut-off distance of 7 A, are -23.43, -22.72, -22.71 and -21.69 kcal mol- ', respectively. They are comparable with those of the deepest minima of the system NaGDC-LBR (-24.0 kcal mol-') and NaGDC-RBR (-22.6 kcal mol- '), calculated with the same cut-off distance." The energy difference in this case is higher than that for NaGC-BR, in agreement with the greater NaGDC enantioselective ability." On the other hand, the helical micellar models of NaGDC and NaDC, as well as their enantioselective ability, are very similar." Hence, the greater enantioselective complexation of BR with NaDC than that with NaGC is justified (see Fig. 7 of ref.8 and Fig. 9 of this work). The similar behaviour of NaGC and NaTC is easily explained by assuming the two structural units observed in the NaTC monoclinic crystal as models for the NaTC micellar aggregates. Previously, their close resem- blance with the 2, helix of NaGC was emphasized.In partic- ular, the region of the NaGC anion more strongly engaged in the formation of the complex with BR includes many atoms of the rings A, B, C and D, and is practically the same in the NaTC anion. From all of these results it seems reasonable to suppose that BR, on the basis of the ellipticity values of its CD spectra, can act as a probe for the recognition of the structural motifs characterizing the bile salt micellar aggre- gates. Further work is in progress to clarify this hypothesis. This work was supported financially by the Italian Consiglio Nazionale delle Ricerche, Progetto Finalizzato Chimica Fine 21 D. Kaplan and G. Navon, Isr. J. Chem., 1983,23, 177. 22 F. R. Trull, J-S.Ma, G. L. Landen and D. A. Lightner, Isr. J. Chem., 1983,23,211. 23 G. Navon, S. Frank and D. Kaplan, J. Chem. Soc., Perkin Trans. 2, 1984, 1145. 24 G. Blauer and G. Wagniere, J. Am. Chem. SOC., 1975,97, 1949. 25 Y-M. Pu and D. A. Lightner, Croat. Chem. Acta, 1989,62,301. 26 J. H. Perrin and M. Wilsey, J. Chem. SOC.,Chem. Commun., 1971, 769. 27 M. Reisinger and D. A. Lightner, J. Inclusion Phenom., 1985, 3, 479. 28 J. Lon Pope, J. Lipid Res., 1967, 8, 146. 29 M. C. Carey, in Sterols and Bile Acids, ed. H.Danielsson and J. Sjavall, Elsevier/North-Holland Biomedical Press, Amsterdam, 1985, ch. 13, p. 380. 30 J. P. Kratohvil, W. P. Hsu, M. A. Jacobs, T. M. Aminabhavi and Y. Mukunoki, Colloid Polym. Sci., 1983, 261, 781. 31 P. Main, S.J. Fiske, S. E. Hull, L. Lessinger, G. Germain, J. P. Declercq and M. M. Woolfson, MULTAN 80. A System of Computer Programs for the Automatic Solution of Crystal Struc- tures from X-Ray Difraction Data, Universities of York (England) and Louvain (Belgium), 1980. 32 M. Camalli, D. Capitani, G. Cascarano, S. Cerrini, C. Giaco- vazzo and R. Spagna, SIR-CAOS: User Guide, Instituto di Strutturistica Chimica CNR C. P. n. 10, 00016 Monterotondo Stazione, Roma, 1986. 33 International Tables for X-Ray Crystallography, The Kynoch Press, Birmingham, 1974, vol. IV. 34 W. Klyne and V. Prelog, Experientia, 1960, 16,521. 35 C. Altona, H. J. Geise and C. Romers, Tetrahedron, 1968,24, 13. 36 E. Giglio and C. Quagliata, Acta Crystallogr., Sect. B, 1975, 31, 743. 1532 J. CHEM. SOC. FARADAY TRANS., 1994, VOL. 90 37 38 39 E. Giglio, in Inclusion Compounds, ed. J. L. Atwood, J. E. D. Davies and D. D. MacNicol, Academic Press, London, 1984, vol. 2, p. 207. M. DAlagni, A. A. DArchivio and E. Giglio, Biopolymers, 1993, 33, 1553. R. Brodersen, Acta Chem. Scand., 1966,20,2895. 41 42 43 M. C. Carey and A. P. Koretsky, Biochem. J., 1979,179,675. N. G. Parsonage and R. C. Pemberton, Trans. Faraday Soc., 1967,63, 311. B. R. Brooks, R. E. Bruccoleri, B. D. Oldson, D. J. States, S. Swiminathan and M. Karplus, J. Comput. Chem., 1983,4,187. 40 P. E. Hansen, H. Thiessen and R. Brodersen, Acta Chem. Scand., Part B, 1979,33,281. Paper 3/05912J;Received 1st October, 1993