Speed of Sound, Density, and Molecular Modeling Studieson the Inclusion Complex between Sodium Cholate and

â-Cyclodextrin

Gustavo Gonzalez-Gaitano,† Aurora Compostizo,‡ Luis Sanchez-Martın,§ andGloria Tardajos*,‡

Departamento de Quımica-Fısica I, Facultad de Ciencias Quımicas, Universidad Complutensede Madrid, 28040 Madrid, Spain, and Departamento de Quımica Organica, Facultad de

Ciencias Quımicas, Universidad Complutense de Madrid, 28040 Madrid, Spain

Received August 13, 1996. In Final Form: January 29, 1997X

The system sodium cholate (NaC) + â-cyclodextrin (â-CD) in water has been studied by speed of soundand densitymeasurements to obtain the corresponding apparent and partial molar volumes and adiabaticcompressibilities. For pure NaC the values for the micellization volumes and compressibilities have beenobtained, as well as the transference properties due to the complexation for the ternary system. Whenthe â-CD is present, a shift in the critical micelle concentration of the surfactant equivalent to the amountof â-CD added is observed, due to the complex formation between solutes that delays the micellization.At infinite dilution, there is a marked change in the compressibility of the surfactant, although it is notappreciable in the volume. A detailedmolecularmodeling study has been carried out to elucidate, togetherwith 1H NMR data, the microscopic structure of the complex.

Introduction

â-Cyclodextrin (â-CD) is a cyclic oligosacharideobtainedas themain product from the enzymatic conversion of thestarch. It consistsof sevenR-D-glucopyranoseunits, linkedby glycosidic bonds R-1,4. Due to a lack of free rotationabout the glycosidic bonds, â-CD (as the rest of CDs) hasa unique spatial configuration, showing a cylindrical orhollow truncated cone shape. The cavity,with6Åofwidthand 7.9 Å of height, has a hydrophobic character, whilethe rimsarehydrophilic: thewider one,with14 secondaryhydroxyl groups, and a narrower one, with the 7 primaryOH groups (Figure 1a). These structural features givethe CD the property of forming inclusion complexesbetween molecules that fit into the hydrophobic cavity.There aremany studies dealingwith inclusion complexesof â-cyclodextrin, either in its natural state or withmodified â-CDs, and with a great variety of guestmolecules, that find application in different fields.1

Bile salts are natural amphiphiles which are synthe-sized in the liver. They form small aggregates2 whichhelp to solubilize and disperse dietary lipids. They havea considerable aqueous solubility andalso agreat capacityto solubilize molecules such as lecithin and cholesterol.3Bile salts are also commonly used for membrane solu-bilizationandreconstitution, specially the sodiumcholate,NaC.4 Fromthegroupofbiological surfactants constitutedby bile acids salts, sodium cholate is one of themost used.It is made up (Figure 1b) of a rigid steroid nucleus, in

which the cyclohexanes A and B are in the cis position;there are three hydroxyl groups inserted at positions 3,7, and 12. Position 17 is occupied with a short aliphatictail, with a carboxylate group at the end. Thus, themolecule presents a hydrophobic face (with three methylgroups, C18, C19, and C21), an aliphatic chain finishedin a carboxylate anion, and a hydrophilic face.There are several reports on inclusion complexes

betweenNaCandCDsstudiedby liquid chromatography,5fluorescence,6 microcalorimetry,7 or NMR.8 The aim ofthis workwas to study not only the complex structure butmainly the role that â-CD plays in the micellizationbehavior of the NaC. The study has been performedthrough two partial and apparent molar properties,namely, molar adiabatic compressibility and molar vol-

* Towhomcorrespondence should be addressed: Departamentode Quımica-Fısica I, Facultad de Ciencias Quımicas, UniversidadComplutensedeMadrid, 28040Madrid, Spain.Fax: 34-1-3944135.E-mail: tardajos@eucmax.sim.ucm.es.

† Departamento de Quımica-Fısica I.‡ Departamento de Quımica Organica.X Abstract published inAdvanceACSAbstracts,March15, 1997.(1) (a) Szelti, J. Cyclodextrins and Their Inclusion Complexes;

Akademiai Kiado: Budapest, Hungary, 1982. (b) Bender, M. L.;KomiyamaM.Cyclodextrin Chemistry; Springer-Verlag: Berlin, 1978.(c) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362. (d)Song Li; Purdy, W. C. Chem. Rev. 1992, 92, 1457-1470.

(2) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B.Biochemistry 1979, 18 (14), 3064-3075.

(3) Carey, M. C.; Small, D. M. J. Clin. Invest. 1978, 61, 998.(4) Tauskela, J. S.; Akler, M.; Thompson, M. Anal. Biochem. 1992,

201, 282-287.

(5) Shimada, K.; Oe, T.; Hirose, Y, Komine, Y. J. Cromatogr. 1989,478, 339.

(6) Miyahima, K.; Yokoi, M.; Komatsu, H.; Nakagaki, M. Chem.Pharm. Bull. 1986, 34, 1395.

(7) Tan, X.; Lindembaum, S. Int. J. Pharm. 1991, 74, 127.(8) Tan, Z. J.; Zhu, X. X.; Brown, G. R. Langmuir 1994, 10, 1034.

a

b

Figure 1. (a) â-Cyclodextrin structure (â-CD). (b) Cholate ionstructure.

2235Langmuir 1997, 13, 2235-2241

S0743-7463(96)00803-7 CCC: $14.00 © 1997 American Chemical Society

ume, obtained by very precise measurements of densityand speed of sound. Given the short range of concentra-tions available and the small changes in these properties,it has been necessary to make use of a high-precisiontechnique, designed by us, to obtain simultaneously bothsound velocity and density. Molecular modeling tech-niques and 1HNMR spectroscopy have been employed aswell to elucidate themicroscopic structureof the complexesand to estimate the energies involved in the inclusionprocess.

Experimental SectionMaterials. â-CD was obtained from Aldrich and NaC from

Sigma. The purity of the â-CD was 99.5% and that for NaC isbetter than 98%. The water content of the cyclodextrin wasdetermined by thermogravimetric analysis and was found to be13.5%. All the products were used as received, without furtherpurification. For the NMRmeasurements deuterium oxide wasemployed as solvent, fromMerck, with a deuteration degree notless than 99.95%. Thewater of the â-CDwas taken into accountin the concentration of the solutions, which were prepared byweight.Apparent Molar Properties. Measurements of the speed

of sound and density were performed simultaneously with atechnique designed in this laboratory (Figure 2). The measure-ment of both properties was carried out in a continuous way, asdescribed below, thus permitting higher precision in the partialmolar properties. The part of the technique related to the speedof sound is described extensively inaprevious report.9 It consistsof measuring the time that it takes a wave-packet of definedultrasonic frequency to travel thedistancebetweenapiezoelectrictransducer and a reflector. A digitizing oscilloscope of highsampling rate (20 MS s-1) counts the time between twoconsecutive reflectionsand the speedof sound is obtaineddirectlyfrom the travel time of the wave-packet and the known distancetransducer-reflector,which is calibrated in eachexperimentwitha reference liquid (pure water). The ultrasonic cell is immersedin a thermostat, controlled by a TRONACPTC41 and a cryostat.The stability in the temperature is better than 1 mK (all theexperiments were carried at 298.15 K). The densities weremeasured with a vibrating tube densimeter Anton Paar DMA601 HT, calibrated with water and air in each set of measure-ments, and thermostated by recirculation of the water of thethermostat (there is no temperature difference between thesolution in the densimeter and in the cell). A peristaltic pumpsuctions liquid to the densimeter from the ultrasonic cell, whereamagnetic stirrer produces, together with the recirculation, theadequatehomogenizationof themedium. Whenthe recirculationis judged tobecompleted, thepumpis turnedoff, and thevibrationperiod of the densimeter and the time of flight of thewave-packet

are registered. The complete process of stirringand stabilizationof the measurements use to take about 15 min.After the measurement, an automatic buret, Metrohm 665,

changes the concentration in the cell by adding volumes of amore concentrated solution, and the process of recirculation isrepeated. A PC controls both the oscilloscope and the buret andit is programmed to follow the evolution of the wave-packetswith the concentration. It calculates the volumes to be added,to keep constant the increments of concentration, and takes themeasurements of added volume, time, and period. Measureddata are stored on the hard disk and sent to the printer. Thedensimeter, the peristaltic pump, and the buret are containedin a polymethacrylate box at 25 ( 0.05 °C, to avoid temperaturegradients. In these conditions, precision in speed of sound anddensity are 2 × 10-3 m s-1 and 1 × 10-6 g cm-3, respectively.In all the experiments a stock solution was prepared by

weighing an amount of â-CD in water. Part of this solution wasintroduced into the measuring cell, and with the rest anotherwas prepared with the desired molality of NaC. The latter isfilling theburetand it is added to theultrasonic cell. Thispermitsvariationof the surfactantmolalitykeeping constant themolalityof â-CD.Molecular Modeling. The calculations were done with the

Insight II program,10 implemented in an IRIS 4D/310VGXworkstation of Silicon Graphics. Themolecular structure of theâ-CD has been generated by linking of seven units of R-D-glucopyranose, and that of the cholate ionbymeans of theSketchutility from themainmodule Builder. Energyminimizations ofthe isolated host and guest molecules were performed with theDiscover program, employing the CVFF forcefield,11 and withseveral algorithms (at first a steepest descents algorithm,finishing with a modified Newton-Raphson to refine thestructures), until the root mean squares of the derivatives wereless than 0.0001 kcal Å-1. Afterward, molecular dynamiccalculations of the system at 298 K were performed, and theprocess of minimization was repeated, to find the absoluteminimum of energy. The structure of the CD thus obtained ishighly regular (the O4 atoms are almost coplanar), more thanthe crystalline structure that is obtained fromX-rayandneutrondiffraction data available at the Cambridge Structural DataBase.12 We have chosen this highly symmetric structure ratherthan the crystalline one, which is slightly distorted because ofthe hydration water that is inside and outside the cavity.To fit the cholate anion into the cavity, rigid docking experi-

ments were carried out with the refined structures. The cholatewas approximated by the steroid nucleus and by the aliphatictail, anddirected toward the two rimsof theCD,until thepositionthat gives a minimum interaction energy was found. Thispositionwas takenas the startingpoint tominimize the complex.The algorithm employed in theminimization of these complexeswas a conjugate gradients, until the rootmean squareswere lessthan 0.001 kcal Å-1. The cut-off distances for the Coulomb andvan derWaals energies were of 100 Å, to include all the possibleinteractions. The association energies were obtained by sub-tracting the complex energy from that of the CD and the cholateion. Cross-terms and harmonic potentials for bonding energieswere included in the force field. To account for solvent effects,a relative dielectric constant of 80 dependent on inverse of thedistance was introduced into the Coulombic term of the forcefield. A common strategy usually followedwhenmodeling theseCD complexes is to assume that the molecules are in a vacuum.However, it is known that for these complexes solvent plays adecisive part, so going without it could be a rather drasticapproximation when energies are estimated or geometries arepredicted.NMRMeasurements. The samples forNMRwere prepared

inD2Oas solvent, at severalmolar ratiosNaC:â-CD, andkeepingfixed the â-CD concentration at 0.015M. TheNMRspectrawererecorded in a Varian VXR 300S, at 300 MHz, fitted with athermostating unit. All the records were carried out at 20( 0.4°C. The chemical shifts are relative to the HDO signal, at 4.63ppm.

(9) Gonzalez-Gaitano,G.; Tardajos,G.;Montero deEspinosa, F.Rev.Sci. Instrum. 1994, 65, 2933.

(10) Insight II version 2.1.0. SanDiego: BiosymTechnologies, 1992.(11) Dauber-Osguthorpe, P.; Roberts, V.A.;Osguthorpe,D. J.;Wolff,

J. Proteins: Struct., Funct., Genet. 1988, 4 (1), 31-47.(12) CSD,CambridgeCrystallographicDataCentre,Cambridge,U.K.

Figure 2. Experimental setup.

2236 Langmuir, Vol. 13, No. 8, 1997 Gonzalez-Gaitano et al.

Results and DiscussionApparent and Partial Molar Properties. The

apparent molar volume and apparent molar adiabaticcompressibility of a solute i inamixturewhere themolalityof the other components is kept constant canbe calculatedusing the usual definitions:

where âS ) (Fu2)-1 is the adiabatic compressibility, andthe zero subindex stands for the initial state when themolalitymi is zero. In this paper the molality is definedasmoles of solute per kilogram of solvent according to theIUPAC, where the solvent, w, is pure water. Accordingto theabovedefinitionofmolality the resultingexpressionsfor the apparent molar properties are

F is the density of the solution, F0 the density whenmi )0,Mi andMj are the molar mass of solutes i and j, andmiand mj the molalities. The partial molar properties canbe obtained from the apparent ones by using

It is possible to define another molality scale, where themolality of i would be moles of i per kilogram of mixedsolvent w + j, although in this case, in which i and j aresolid solutes it is more appropriate to use the IUPACdefinition of molality. Anyway the conversion betweenboth scales is straightforward and, for dilute systems,differences are minimal. The difference between eqs 3and 4 and those usually found in the literature is thatmj(the molality of the third component) does not appear inthe above expressions.

â-Cyclodextrin. The density and ultrasonic velocityfor â-CD solutions in water as a function of the molalityare plotted in Figure 3. The apparentmolar volumes andcompressibilities were calculated according to eqs 3 and4, and they are represented in Figure 4 together withvalues from the literature.13-15 The experimental pointswere fitted to the following equations:

where the superindex zero stands for the corresponding

properties at infinite dilution (standard state) and the Bcoefficients account for the solute-solute interactions.Our results for the volumes are in good agreementwith

the literaturedata, especiallywith those ofMilioto et al.,14although they obtained a slightly higher value at infinitedilution (706.5 cm3 mol-1) and a negative Bv coefficient(-95 cm3 mol-2 kg). A negative value is indicative of anhydrophobic interaction, something which lacks consis-tency with the 21 hydroxyl groups of the â-CD. With

(13) Paduano, L.; Sartorio, R.; Vitagliano, V.; Constantino, L. J.Solution Chem. 1990, 19, 31.

(14) Milioto, S.; Bakshi, M. S.; Crisantino, R.; De Lisi, R. J. SolutionChem. 1995, 24, 103.

(15) Nomura, H.; Koda, S.; Matsumoto, K.; Miyahara, Y. Studies inPhysical and Theoretical Chemistry; Elsevier Science Publishers:Amsterdam, 1982; Vol. 27, p 151.

vφ,i ) (V - V0)/mi (1)

κS,φ,i ) - 1mi

{(∂V∂P)S

- (∂V0

∂P )S} ) 1

mi(âSV - âS,0V0) (2)

vφ,j ) Mi/F - (1 + mjMj)(F - F0)/miFF0 (3)

κS,φ,i ) âSvφ,i + (1 + mjMj)(âS - âS,0)/miF0 (4)

vi ) (∂V∂ni)nw,nj ) ddmi

(vφ,imi) (5)

κS,i ) -( ∂∂ni(∂V∂P)S)nw,nj

) ddmi

(κS,φ,imi) (6)

vφ,CD ) v0CD + BvmCD )

704.04 + 17mCD (cm3 mol-1)

κS,φ,CD ) κ0S,CD + Bκ,SmCD ) -2.59 × 10-15 +

0.145 × 10-12 mCD (Pa-1 m3 mol-1)

Figure 3. (a) Density measurements and (b) speed of soundversusmolality for pure substances and â-CD+NaCmixtures(fixed concentrations of â-CD 0.008 44 m and 0.012 14 m).

Inclusion Complex Studies Langmuir, Vol. 13, No. 8, 1997 2237

reference to the adiabatic compressibility, the low ex-trapolated value (only slightly negative, -2.59 × 10-15

Pa-1m3 mol-1) agrees well with Nomura et al. data,15showing, as in the volume, a positive slope. The smallvalue of the standard compressibility suggests a strongreorganization of the hydration water of the â-CD.SodiumCholate. Theresults ofdensityandultrasonic

velocity for NaC are shown in Figure 3. Althoughdeviations of the density from a single straight line arenegligible, on the u graph it is possible to plot two lineswith different slopes that intercept at 0.015 m thatcorresponds to the critical micelle concentration (cmc).16With this technique it has been possible to make enoughmeasurements below the cmc and to obtain the partialmolar volumes and compressibilities of NaC at infinitedilution. The scattering in the plots of partial molarproperties is in part a consequence of the direct derivativeover theexperimental values,withoutanyprevious fitting.The calculated volumes obtained according to eqs 3 and

5 are plotted in Figure 5. At infinite dilution vNaC is 322.7cm3 mol-1. The micellization volume, defined as thedifference of the molar partial volume of the surfactantinmicellized form(plateauregion)and inmonomeric form,is ∆vM ) 6.4 cm3 mol-1. This value is similar to thatobtained for ionic surfactantswith a long alkyl chain (e.g.,7 cm3 mol-1 for dodecyltrimethylammonium bromide17)but the shape of the curve is different: the range ofconcentration needed to reach a stationary value afterthe beginning of the aggregation is larger for the NaC.Such a broad micellar region indicates a multistepaggregation process in which species of different aggrega-tion number are involved, as reported by Djavanbakht etal., based onmeasurementsusingultrasonic absorption.18

In Figure 6 are plotted the apparent and partial molarcompressibilities. These data are less noisy than thevolumes,because therelativechanges in thespeedof soundare larger than those of the density. The above commentsrelated to the nature of the aggregation process are morenoticeable in thisproperty. Althoughobserving this figurewe can appreciate that κS does not reach an absolutelyconstant value,whichwould be a clear indication that theaggregation continues well above the initial aggregationpoint, we can give an estimated value of themicellizationcompressibility,∆κSM, of 7.2×10-14 Pa-1m3mol (obtainedin the same way as ∆vM), a value that falls within thesame order of many surfactants in water.19,20 Theadiabatic molar compressibility of the surfactant in itsmicellized form increases along a broad region from a lownegative value to another close to zero. The low value ofthe compressibility of theNaC in themicelles is indicativeof a persistence of the interaction via hydroxyl groupswith the solvent and/or between the cholate ions in theaggregates, in keeping with Small’s model for the ag-gregation of bile salts.21 Despite the low cmc concentra-tions it is possible to obtain with accuracy the compress-ibility of the monomer at infinite dilution, resulting in-8.1 × 10-14 Pa-1 m3 mol-1 (see Figure 6).Ternary System. In parts a and b of Figure 3 the

results of density and velocity are shown respectively fortwo different â-CDmolalities (0.00844m and 0.01214m).Given the straight line appearance of both plots, we haveobtained the derivative of the speed of sound data withrespect to the molality of NaC to detect changes in theslope. Such values are shown in Figure 7, and given thatthe derivatives are obtained at moles of water and â-CDconstant, they just represent the change in u per mole ofNaC added. The use of derivatives in the speed of soundto detect structural changes as a function of the temper-ature has been used by Glatter,22 but to our knowledgethere are no studies on the derivative with the concentra-tion. Regarding the pure cholate in themonomeric form,we can observe an initial linear zone that extrapolates ata value of 290 m s-1 mol-1kg, and with a negative slopethat should be a consequence of all the interactions. Asthis is a differential property, it is easier to see the

(16) O’Connor, C. J.; Wallace, R. G. Adv. Colloid Interface Sci. 1985,22, 1-111.

(17) Guvelli, D. E.; Kayes, J. B.; Davies, S. S. J. Colloid Interface Sci.1981, 82, 307.

(18) Djavanbakht, A.; Kale, M.; Zana, R. J. Colloid. Inerface Sci.1977, 59, 139.

(19) De Lisi, R.; Ostiguy, C.; Perron, G.; Desnoyers, J. E. J. ColloidInterface Sci. 1979, 71 (1), 147.

(20) Vikingstad, E.; Skauge, A.; Høiland, H. J. Colloid Interface Sci.1978, 66 (2), 240.

(21) Small, D.M. InThe bile acids; Nair, P. P., Kritchevsky, D., Eds.;Plenum Press: New York, 1971; Vol 1, Chapter 8.

(22) Glatter, O. J. Phys. IV 1995, 3, 27.

Figure 4. Apparent molar volumes and compressibilities forâ-CD.

Figure 5. Apparent and partial molar volumes for pure NaC.

2238 Langmuir, Vol. 13, No. 8, 1997 Gonzalez-Gaitano et al.

beginning of the aggregation than with the u plot. Thus,whereas micelles start to form at 0.011m, the maximumchange in the slope of the derivative (second derivative)is around 0.015 m, corresponding to the value obtainedat the intercept of two straight lines in u.The values at infinite dilution of (∂u/∂mNaC)w,â-CD are

different than those in pure water and, given that theproperty represents the change in u per mole of addedNaC at fixed CD concentration, providing evidence of aninteraction between the NaC and the â-CD. Theseextrapolations are the same (251 m s-1 kg mol-1) withindependence of the â-CD concentration. At infinitedilution there is an excess of CDwith respect to the NaC,and mNaC/mCD ) 0. Regardless of the value of theequilibrium constant for the complexation, in such condi-tions all the surfactantwill be in complexed form, and thevalue of the properties at infinite dilution must beindependent of the fixedCD concentration. Although thedifferent values of the (∂u/∂mNaC) confirm the interactionbetween NaC and CD, it is not possible to observe a neatchange in the plots corresponding to the 1:1 complex.Nevertheless, it is possible to observe a shift on the cmcof the plots to higher concentrations, in an amount equalto the added â-CD. This confirms the formation of acomplex of 1:1 stoichiometry, which is strong enough tomodify themicellization equilibrium. Another conclusionthat can be drawn from these results is that, provided thevalues of (∂u/∂mNaC)w,â-CD are the same but shifted to anewmolality,m*)mNaC +mâ-CD, the â-CD is not formingpart of the aggregates. In view of the specificity of the

geometric and polarity requirements for the formation ofbile salt aggregates,21 it would be difficult for the â-CD totake part in the formation of the micelles.Similar conclusions can be obtained using the more

familiar compressibilities. In Figure 8 the apparent andpartial molar compressibilities are plotted. The shift inthe cmc in the presence of â-CD is, of course, more neatin the molar partial compressibility than in the apparentone. For the pure cholate in water the values at infinitedilution are different from those in the systems withcyclodextrin, yielding the same value with independenceof the molality of â-CD. The transfer compressibility,∆κ0S,comp from the pure water to the complex is 2.25 ×10-14Pa-1 cm3mol-1. Thispositivevalue canbeassociatedwith the water molecules included in the cavity of theâ-CD, displaced by the cholate and now forming part ofthe bulk.Apparent molar volumes, vφ,NaC, have also been calcu-

lated. The cholate shows the same volume in the absenceor in the presence of cyclodextrinwithin the experimentaluncertainty. The fact that the transfer volume is practi-cally zero proves that the volume occupied by a cholatemolecule in water is equivalent to the volume of waterdisplaced from the cavity of the â-CD and now formingpart of the bulk water.MolecularModeling andNMR. Figure 9 shows the

structures obtained by molecular modeling according tothemethods described in the Experimental Section. Thecalculated energy values are -37.51 (structure 1) and-37.25 kJ mol-1 (structure 2), practically the same. Ifthe cholate is introduced by the steroid nucleus (seestructures 3 and 4), energies are of-32.08 and-33.16 kJ

Figure 6. Apparent and partial molar compressibilities forpure NaC.

Figure 7. Derivatives with the concentration of NaC for pureNaC and mixtures. Symbols are the same those in Figure 3a.

Figure 8. (a) Apparentmolar compressibilities and (b) partialmolar compressibilities for the NaC + â-CD system.

Inclusion Complex Studies Langmuir, Vol. 13, No. 8, 1997 2239

mol-1, depending if it enters by thenarrower rim (primaryhydroxyl groups) or by thewider rim (secondary hydroxylgroups).From the NMRmeasurements, in the case of the â-CD

one can identify the peaks that correspond to the six typesof protons bonded toC.23 WhenNaC is present, anupfieldshift of H5 may be observed, while H3 and H6 shiftdownfield to a less extent. H1, H2, and H4, that is, theprotons located in the outer face of the cavity, are scarcelyshifted. These results show that the complexation hastaken place. From the plot of ∆δ values against thequotients NaC:â-CD and â-CD:NaC for several R values(Figure 10) a 1:1 complex stoichiometry is evident (∆δ isthe chemical shift minus the value in absence of host orguest molecule). For the cholate protons (notation withapostrophes to distinguish from theCDprotons), we havechosen themost representativeandresolved resonances,24that is, H′3, H′12, H′18, H′19, and H′21 (doublet). Themost affected protons are H′18 and H′21 and, to a lessextent, H′12 and H′19. H′3 does not shift within theexperimental error. Chemical shifts areproducedupfield,in the same direction as H5 of the CD. In general terms,the chemical shifts of the aliphatic chain protons H′18andH′21aregreater thantherest. This isunderstandablein light of the cholate structure: these are protonsbelonging to methyl groups inserted in the hydrophobicside and they will interact much more with the cavity.Methyl protons of the C19 are less affected, meaning thatit is undergoing less interaction, although this is a groupasbulkyas the others. On the otherhand, theH′3, located

at the extremity of the steroid nucleus, do not undergochanges. Accordingly,wemaysay that the cholate is goingto enter into the cavity by the aliphatic chain, that is, bythe carboxylate group. These results are the same thatthose obtained by Tan et al.,7 except in the sense in whichH3protons are shifted (these authors obtainahigher shiftand in the opposite sense). Nevertheless, this fact doesnot change the conclusions.According to the theoretical calculations, any of the first

two structures would be possible, since they are almostequal energetically. The fact that H5 of the CD is theproton that suffers a greatest downfield shift suggests astronger interaction with the cholate (possibly van derWaals shifting or steric perturbation) that should have acorresponding shift in the same direction (if this were thenature of the interaction) in some cholate proton. Thegreatest displacements are H′18 and H′21. This, in viewof the two structures, would be more compatible withstructure 2, in which H5 protons are closer to thehydrogens of C21 and C18, since this is the narrow partof the cavity (cholate is bogged down, like a funnel). H3shifts less indicating aminor contact (wider side) fact thatis more reasonable if the cholate enters by the primaryhydroxyl border, in light of theminimized structures.Tan8suggests that the complex forms when the anionic grouppenetrates the cavity by the secondary rim. Anyway, thecarboxylate group is outside the cyclodextrin, as was tobe expected (in general, ions have little trend to remainincluded in the CDs cavity25) and any of these twocalculated structures is compatiblewith the experimental

(23) Demarco, P. V.; Thakkar, A. L. Chem. Commun. 1970, 2.(24) Campredon,M.; Quiroa, V.; Thevand, A.; Allouche, A.; Pouzard,

G. Magn. Reson. Chem. 1986, 24, 624-629.(25) Hersey, A.; Robinson, B. H.; Kelly, H. C. J. Chem. Soc., Faraday

Trans. 1 1986, 82, 1271-1287.

a

b

Figure 9. Minimized structures for the complex when (a) thecholate enters by the aliphatic chain and (b) the cholate entersby the steroid nucleus. (Only the protons H3 and H5 of theâ-CD and H′3, H′12, H′18, H′19, and H′21 of the cholate aredrawn in the structures.) Figure 10. (a) Chemical shifts of the â-CD protons versus the

molar ratio R and (b) for several protons of the NaC.

2240 Langmuir, Vol. 13, No. 8, 1997 Gonzalez-Gaitano et al.

results. Regarding structures 3 and 4, besides givinghigherenergies, theyarenot consistentwithNMRspectra,since the strong chemical shifts of the resonances of H′18and H′21 should not be observed (because they would beoutside the CD) and a change on the chemical shift of H′3should be expected. The carboxylate group goes throughthe cavity remaining hydrated, so there is no drawbackto the cholate ion to enter by the charged group.

∆δ versus R plots permit us to estimate the bindingconstant K by application of the Benesi-Hildebrandmethod, assuming a 1:1 complex. We have obtained anequilibrium constant ofK) 2200( 400M-1 using theH5proton. For H′18 the calculated value was of 3400 ( 300M-1. The results are in good agreement with the valuesof the literature obtained by NMR8 (1500 M-1, only with

the protons H′18) andmicrocalorimetry7 (3150M-1). Thevalue is typical of surfactant guests, and it is high enoughto resolve the competitive equilibrium between micelli-zation and complexation in favor of the latter, so a changein the cmc is expected in an amount equal to that of theconcentration of CD, as observed in the apparent molarproperties, since the complex is 1:1.

Acknowledgment. We are grateful to the M.E.C. ofSpain for financial support through twoDGICYTGrants,PB89-0113 and PB-930448, to the Centro de Espectrosco-pıa, and to the Vicerrectorado de Investigacion of theU.C.M. for the postgraduate grant for G.G.

LA960803T

Inclusion Complex Studies Langmuir, Vol. 13, No. 8, 1997 2241