DOI: 10.1021/la103124n 17949Langmuir 2010, 26(23), 17949–17957 Published on Web 11/03/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Hydroxypropyl-Substituted β-Cyclodextrins: Influence of Degree of

Substitution on the Thermodynamics of Complexation with Tauroconjugated

and Glycoconjugated Bile Salts

Christian Sch€onbeck,†,‡ Peter Westh,† Jens Christian Madsen,§ Kim Lambertsen Larsen,z

Lars Wagner St€ade,z and Ren�e Holm*,‡

†NSM, Research Unit for Functional Biomaterials, Roskilde University, Universitetsvej 1, DK-4000 Roskilde,Denmark, ‡Preformulation, H.Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark, §Compound ID &

Purification, H.Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark, and zSection of Chemistry,Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University,

Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark

Received August 5, 2010. Revised Manuscript Received September 30, 2010

The effect of the degree of substitution (DS) on the ability of hydroxypropylated β-cyclodextrin (HPβCD) to forminclusion complexes with six different bile salts, found within the intestinal tracts of rats, dogs, and humans, was studiedby isothermal titration calorimetry. The composition and molecular structure of the cyclodextrin samples werecharacterized by MALDI-TOF mass spectrometry together with 1D and 2D-NMR, and some of the complexes werestudied by 2D ROESY NMR. The stability and structure of the complexes were mainly determined by the position ofhydroxyl groups on the bile salts and depended relatively little on the number of hydroxypropyl side chains on the CDs.The enthalpy and entropy of complexation exhibited a strong linear increase as the DS increased from 0 to 1, and apronounced enthalpy-entropy compensation was observed. These observations are interpreted as an increased releaseof ordered water from the hydration shells of the bile salts, caused by the hydroxypropyl substituents on the rim of theCD. It is estimated that each CD hydroxypropyl substituent dehydrates a hydrophobic surface area of approximately 10 A2.

Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides with the shapeof a truncated cone. Owing to their hydrophilic outer surface andtheir lipophilic cavity, they are able to form inclusion complexeswith hydrophobic guests in aqueous solution. Thus, they may actas solubilizers of water insoluble drugs and are used as drugcarriers among other things.1-13

The most common CDs are R, β, and γ-cyclodextrins, consist-ing of six, seven, and eight glycopyranose units, respectively. Thesize of the cavity increases with the number of glucopyranoseunits, and this has a large influence on their complexation capa-bilities, since a strong interaction requires a good fit between theguestmolecule and theCD cavity.14 Thus, there is a preference forthe inclusion of guest molecules or parts of guest molecules thatmatch the size of the cavity.

βCDhas a relatively low aqueous solubility that severely limitsits uses. Substituting the hydroxyl groups at the rim of the βCDresults in a dramatic increase in aqueous solubility, which mightbe due to interruption of intramolecular hydrogen bonds betweenhydroxyl groups on the rims of the βCD.15 The high solubility of2-hydroxypropylated βCD (HPβCD) makes it a good alternativeto the natural βCD, and it is used in several marketed pharma-ceutical products.3,4

However, modifications of the natural CDs not only alter thephysicochemical properties of the CDs and their complexes, butalso the complexation abilities of the CDs.16-22 A number ofstudies have investigated how the stability constants of thecomplexes are affected by the degree of substitution (DS) ofHPβCD.17-21 This strongly depends on the type of guest. Forsome guests, the stability constant increases with increasing DS,while for others it decreases. Generally, “large spherical” guests,exemplified by phenolphthalein, form weaker complexes withincreasing DS, while smaller guests form stronger complexes asthe DS increases.19,20 The substituents are thought to affectcomplexation in two ways: (1) steric blockage of the cavityopenings by the substituents, resulting in a lower stability con-stant and (2) extension of the CD cavity, leading to increasedhydrophobic interactions with the hydrophobic parts of the guest

*To whom correspondence should be addressed. Email: rhol@lundbeck.com. Tel.: (þ45) 3643 3596. Fax: (þ45) 3643 8242. Address: H.LundbeckA/S,Ottiliavej 9, DK-2500 Valby, Denmark(1) Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98, 2045–2076.(2) Carrier, R. L.; Miller, L. A.; Ahmed, I. J. Controlled Release 2007, 123,

78–99.(3) Davis, M. E.; Brewster, M. E. Nat. Rev. Drug Discovery 2004, 3, 1023–1035.(4) Brewster, M. E.; Loftsson, T. Adv. Drug Delivery Rev. 2007, 59, 645–666.(5) Loftsson, T.; Brewster, M. E.; Masson, M. Am. J. Drug Delivery 2004, 2,

175–261.(6) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht,

The Netherlands, 1988.(7) Szejtli, J. Med. Res. Rev. 1994, 14, 353–386.(8) Rajewski, R. A.; Stella, V. J. J. Pharm. Sci. 1996, 85, 1142–1169.(9) Uekama, K.; Otagiri, M. Crit. Rev. Ther. Drug 1987, 3, 1–40.(10) Challa, R.; Ahuja, A.; Ali, J.; Khar, R. K. AAPS PharmSciTech 2005, 6,

E329–E357.(11) Arima, H.; Hirayama, F.; Okamoto, C. T.; Uekama, K. Recent Res. Devel.

Chem. Pharm. 2002, 2, 155–193.(12) Thompson, D. O. Crit. Rev. Ther. Drug 1997, 14, 1–104.(13) Uekama, K. Chem. Pharm. Bull. 2004, 52, 900–915.(14) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875–1917.

(15) Saenger,W.; Betzel, C.; Hingertu, B.; Brown,G.M.Angew. Chem. 1983, 95,883–884.

(16) Proniuk, S.; Blanchard, J. J. Pharm. Sci. 2001, 90, 1086–1090.(17) M€ullers, B. W.; Brauns, U. J. Pharm. Sci. 1986, 75, 571–572.(18) Zia, V.; Rajewski, R. A.; Stella, V. J. Pharm. Res. 2000, 17, 936–941.(19) Yuan, C.; Jin, Z. Y.; Li, X. H. Food Chem. 2008, 106, 50–55.(20) Buvari-Barcza, A.; Barcza, L. Talanta 1999, 49, 577–585.(21) Rao., C. T.; Pitha, J.; Lindberg, B.; Lindberg, J.Carbohydr. Res. 1992, 223,

99–107.(22) Holm, R.; Madsen, J. C.; Shi, W.; Larsen, K. L.; St€ade, L. W.; Westh, P.

J. Incl. Phenom. Macrocycl. Chem. Published online DOI: 10.1007/s10847-010-9831-3.

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Article Sch€onbeck et al.

molecule, resulting in a higher stability constant.12Not only is thedegree of substitution important but the pattern of substitutionalso influences complexation properties.19,20 As sketched inFigure 1, each of the glycopyranose units may be substitutedat three different sites: O2, O3 and O6. O2 and O3 are situated onthe rim of the wider opening of the CD, while O6 is on the rim ofthe narrow opening. It is speculated that the presence of substit-uents at O2 and O6 may extend the hydrophobic cavity, whereassubstituents at O3 and O6 may narrow the cavity openings12 andmolecular dynamics simulations of HPβCD in water show thatHP groups at the O2 sites expand the wider cavity opening.23

While several authors have investigated the influence of the DSon the stability constant,K, only a few studies have systematicallyinvestigated howvariations in theDSaffect other thermodynamicparameters.18,24-26 In those studies, the stability constants ofcomplexes formed between various guest molecules and sulfo-butylether-βCDs and HPβCDs of different DS were obtained, aswell as the thermodynamic parametersΔH� andΔS� for complexformation. Knowing the latter two parameters allows for a moredetailed interpretation of the role that the substituents play in thestability of the complexes and a better understanding of thedifferent contributions to the stability of the complexes.

In the present study, six bile salts (BS) (Figure 2) that arepresent in the bile of man, rat, and dog27 were employed as guestmolecules for complexation with HPβCD. Bile salts and theirinteraction with CDs are of interest as they bind competitively todrug:CD complexes in the small intestine, thereby releasing thedrug.13,28,29 For this reason, it is important to understand howchanges in the DS affect the strength of CD:BS complexes.Complexes between CDs and bile salts have already been studied

by a range of experimental techniques;30-43 however, no system-atic investigations exist on the influence of CD substitution on thethermodynamics of the interaction.

The purpose of the present study is, therefore, to examine thethermodynamics of the interaction between bile salts andHPβCDs in order to elucidate the effects of HP-substitutionsand to obtain a more general understanding of the driving forcesbehind the inclusion complex formation between cyclodextrinsand hydrophobic guest molecules. This may assist in a rationalchoice of modified βCD for oral drug delivery.

Experimental Section

Materials. Sodium salts of the bile acids taurocholate (TC)(2-([3R,7R,12R-trihydroxy-24-oxo-5β-cholan-24-yl]amino)-ethanesulfonic acid), taurodeoxycholate (TDC) (2-([3R,12R-dihydroxy-24-oxo-5β-cholan-24-yl]amino)ethanesulfonic acid),taurochenodeoxycholate (TCDC) (2-([3R,7R-dihydroxy-24-oxo-5β-cholan-24-yl]amino)ethanesulfonic acid), glycocholate (GC)(3R,7R,12R-trihydroxy-5β-cholan-24-oic acid N-(carboxymethyl)-amide), glycodeoxycholate (GDC) (3R,12R-dihydroxy-5β-cholan-24-oic acid N-(carboxymethyl)amide) and glycochenodeoxycholate(GCDC) (3R,7R-dihydroxy-24-oxo-5β-cholan-24-oic acidN-(carboxy-methyl)amide) were purchased from Sigma-Aldrich (St. Louis,MO). Samples of HPβCDs having different average DS werepurchased from Sigma-Aldrich and CycloLab (Budapest,Hungary). All other chemicals were of analytical grade or higherand milli-Q water was used for the solutions. All chemicals wereused without further purification.

Mass Spectroscopy.MALDI-TOFMSwas performed usinga fast evaporating nitro-cellulose (FENC) matrix on a Reflex IIIinstrument (BrukerDaltonics, Bremen,Germany).A thin layer offreshly prepared saturated R-cyano-4-hydroxycinnamic acid(CCA) in acetone was deposited on a MALDI target plate andallowed to dry. The CD was then deposited directly to theMALDI target plate by adding a 1 μL droplet of a 1:1 solutionof the CD (10 mM) and a 1:4 (v/v) mixture of nitro-cellulose andsaturated CCA in an aqueous solution of 0.1% triflouroaceticacid and 80% acetonitrile.

NMR Spectroscopy. The NMR experiments were carriedout at 25 �C on a Bruker Avance-600 NMR (Bruker Biospin,Rheinstetten, Germany) spectrometer operating at 14.1 T andequipped with a cryogenically cooled probe: 32 scans wereacquired for the 1D 1H experiments, 5120 scans were acquiredfor the attached proton test (APT) experiments, while for the two-dimensional HSQC and HMBC experiments, 32 and 64 scans,respectively, were acquired for each of the 128 t1 increments. TheCDsampleswere run at 10mMinD2O.TheROESYspectrawererecorded on samples containing 10 mM CD and 10 mM BS inD2O and 16 scans were acquired for each of the 256 t1 increments.

Figure 1. One of the seven glycopyranose units of substitutedβCD.

(23) Yong, C. W.; Washington, C.; Smith, W. Pharm. Res. 2008, 25, 1092–1099.(24) Castronuovo, G.; Niccoli, M. Bioorg. Med. Chem. 2006, 14, 3883–3887.(25) Castronuovo, G.; Niccoli, M.; Varriale, L. Tetrahedron 2007, 63, 7047–

7052.(26) Terekhova, I. V.; Obukhova, N. A.; Agafonov, A. V.; Kurochkina, G. I.;

Syrtsev, A. N.; Gratchev, M. K. Russ. Chem. Bull. 2005, 54, 1883–1886.(27) Alvaro, D.; Cantafore, A.; Attili, A. F.; Gianni Corrandini, S.; De Luca, C.;

Minervini, G.; Di Base, A.; Angelico, M. Comp. Biochem. Physiol. 1986, 83B,551–554.(28) Ono, N.; Hirayama, F.; Arima, H.; Uekama, K.; Rytting, J. H. J. Incl.

Phenom. Macrocycl. Chem. 2003, 44, 93–96.(29) Nakanishi, K.; Masada, M.; Nadia, T.; Miyajima, K. Chem. Pharm. Bull.

1989, 37, 211–214.(30) Tan, X.; Lindenbaum, S. Int. J. Pharm. 1991, 74, 127–135.(31) Holm, R.; Nicolajsen, H. V.; Hartvig, R. A.; Westh, P.; Østergaard, J.

Electrophoresis 2007, 28, 3745–3752.

(32) Holm, R.; Hartvig, R. A.; Nicolajsen, H. V.; Westh, P.; Østergaard, J.J. Incl. Phenom. Macrocycl. Chem. 2008, 61, 161–169.

(33) Tan, Z. J.; Zhu, X. X.; Brown, G. R. Langmuir 1994, 10, 5488–5495.(34) Ollila, F.; Pentik€ainen, O. T.; Forss, S.; Johnson, M. S.; Slotte, J. P.

Langmuir 2001, 17, 7107–7111.(35) Mucci, A.; Vandelli, M. A.; Salvioli, G.; Malmusi, L.; Forni, F.; Schenetti,

L. Supramol. Chem. 1996, 7, 125–127.(36) Mucci, A.; Schenetti, L.; Salvioli, G.; Ventura, P.; Vandelli,M.A.; Forni, F.

J. Incl. Phenom. Macrocycl. Chem. 1996, 26, 233–241.(37) Mucci, A.; Schenetti, L.; Vandelli, M. A.; Ruozi, B.; Salvioli, G.; Forni, F.

Supramol. Chem. 2001, 12, 427–433.(38) Vandelli, M. A.; Salvioli, G.; Mucci, A.; Panini, R.; Malmusi, L.; Forni, F.

Int. J. Pharm. 1995, 118, 77–83.(39) Cabrer, P. R.; Alvarez-Parrilla, E.; Al-Soufi, W.; Meijide, F.; N�u~nez, E. R.;

Tato, J. V. Supramol. Chem. 2003, 15, 33–43.(40) Cabrer, P. R.; Alvarez-Parrilla, E.; Meijide, F.; Seijas, J. A.; N�u~nez, E. R.;

Tato, J. V. Langmuir 1999, 15, 5489–5495.(41) Holm, R.; Shi, W.; Hartvig, R. A.; Askjær, S.; Madsen, J. C.; Westh, P.

Phys. Chem. Chem. Phys. 2009, 11, 5070–5078.(42) Liu, Y.; Zhang, Q.; Guo, D. S.; Zhuang, R. T.; Wang, L. H. Thermochim.

Acta 2008, 470, 108–112.(43) Zhao, Y.; Gu, J.; Chi, S. M.; Yang, Y. C.; Zhu, H. Y.; Wang, Y. F.; Liu,

J. H.; Huang, R. Bull. Korean Chem. Soc. 2008, 29(11), 2119–2124.

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Isothermal TitrationCalorimetry (ITC). Prior topreparingthe solutions for the ITC experiments, the solid powders of theBSs andCDswere dehydrated in vacuumat 55 �C for at least 48h.Immediately after dehydration, the appropriate amounts wereweighed out and the solutions were prepared by dissolving thepowders in a 50 mM phosphate buffer, pH 7.1.

The titrations were performed on aMicrocal VP-ITC titrationmicrocalorimeter (MicroCal, Northhampton, MA) at 25.0 �Cand atmospheric pressure. The syringe was loaded with the CDsolution and titrated into the cell containing the bile salt solution.In all the titration runs the volume of the first injection was 2 μLfollowedby27 injections of 10μL,with at least 240 s between eachinjection. The data point corresponding to the 2 μL injection wasdeleted prior to the nonlinear regression described below. Alltitrations were performed in triplicate. Heats of dilution weremeasured in separate runs by titrating the CD solutions intobuffer solution. The measured heats of dilution were subtractedfrom the titration heats to yield the heats of complexation.

The titrationdatawere fitted toamodelbasedononesetof identical,independentbindingsitesusingMicrocals ITCdataanalysisapplicationfor the Origin software package (version 7.0). The regression param-eters are the stoichiometry, n, the molar enthalpy of complexation,ΔH�, and the stability constant, K. The stability constant and molarenthalpy enabled the calculation of the standard Gibbs free energy ofbinding (ΔG�) and the standard change in entropy (ΔS�) according to

ΔG� ¼ -RT ln KS ¼ ΔH�-TΔS�

whereR is the gas constant and T is the absolute temperature.To obtain suitable titration curves for a precise determination

of the binding parameters, the concentrations of the titrant CDsolutions were in the range of 1.25-20 mM and the BS solutionswere in the range 0.125-2 mM. The higher the stability constantswere, the more diluted were the solutions.

Results and discussion

Characterization ofCyclodextrins.UsingNMRandMAL-DI-TOF MS, the substituted cyclodextrins were characterizedwith respect to the degree of substitution and substitution pattern.From the MS spectra, of which an example is shown in Figure 3and the rest are presented in the Supporting Information, it is seenthat eachof thepurchasedcyclodextrin samplesarehighlypolydisperse.In all samples 6-9 HPβCDs of various DS are symmetrically

distributed around the average DS. From the peak intensities, theaverage degree of substitution can be estimated as

DS ¼P

i

IiDSiP

i

Ii

where Ii denotes the intensity of the ith peak and DSi denotes thedegree of substitution corresponding to the ith peak. The averageDS of the used CD samples were also calculated from the 1H

Figure 2. Names and structure of the six investigated bile salt anions.

Figure 3. Mass spectrum ofHPβCDwithDS=0.63. The peak atm/z=1216.5 corresponds to theNaþ adduct ofHPβCDwith onlyone 2-hydroxypropyl substituent perCD-ring, and the peak atm/z=1681.8 corresponds to the Naþ adduct of HPβCD with nine sub-stituents. Kþ adducts are also present in the spectrum.

Table 1. Names and DS of the Studied HPβCDs As Determined by1H-NMR and MALDI-TOF MS

name DS (1H NMR) DS (MALDI-TOF MS)

HP054 0.54 0.59HP063 0.63 0.67HP082 0.82 0.95HP102 1.02 1.05HP106 1.06 1.09

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NMR by comparing the area of the substituent’s methyl group(Hc) to the area of other protons. The results are listed in Table 1.The DS obtained by MS are slightly larger than those obtainedby NMR. Due to variations in the ionization and vaporizationpotentials among the differently substituted molecules in eachsample, there might be a bias in the DS determined byMS. In thefollowing the DS obtained by NMR will therefore be used.

The substitution patterns were determined from both the 1Dand 2D NMR experiments (all 1D 1H and 13C spectra as well as

representative 2D spectra are shown in the Supporting In-formation). Substitution at O2 gives rise to a downfield shift(∼0.2 ppm) of H1 and H3, as well as an upfield shift (∼2 ppm) ofC1 and C3 and a downfield shift (∼8 ppm) of C2 (see Figure 1 fornumbering of the CD nuclei).37,44 All of these extra peaksresulting from the partial substitution at O2 are observed in the

Figure 4. Partial 1H-13C HMBC spectra of HP054 (A) and HP106 (B) showing the correlation peak between H2sub and Ca (see thenumbering of protons and carbons in Figure 1).

(44) Tongiani, S.; Velde, D. V.; Ozeki, T.; Stella, V. J. J. Pharm. Sci. 2005, 94,2380–2392.

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NMR spectra, proving the existence of substitution at this oxygen.Furthermore, a three-bond coupling between the substituent andthe proton on the substituted C2 appear in the HMBC-spectra(Figure 4), providing additional evidence of O2-substitution. Thepercentage of O2s that are substituted may be quantified from thepeak areas ofH1 andH10 (the C1 proton next to a substitutedC2),as these peaks are well separated. Dividing this percentage by theDSyields the fractionof substituents bound toO2.This reveals thatall of the HPβCDs have 50-60% of the substituents located at O2.

It is not possible, however, to determine if the remaining40-50% of the substituents are located on O3 or O6. Theunsubstituted C6 appears as an easily recognizable negative peakin the APT spectra and substitution at O6 would definitely shiftthis peak significantly downfield (a shift of 8-10 ppm is expectedas observed for C2), but such a peak does not appear in any of thespectra. Similarly, there are no features in the NMR-spectra tosupport substitution at O3. The HMBC spectra only show theabove-mentioned coupling between Ca and H2sub as well as theexpected three-bond coupling between Ca and the HP methylprotons, Hc, but no other couplings with Ca are observed. Theexpected peaks and couplings resulting from substitution at O3and/or O6 are probably too broad and too weak to be observed.2D ROESY NMR. A structural characterization of selected

complexes was made using 2DROESYNMR. Twoweakly bind-ing complexes (HP054 and HP106 with GC) and two stronglybinding complexes (HP054 and HP106 with GCDC) wereinvestigated. The assignments of the spectra were made from theHSQC and HMBC spectra of the complexes, and parts of theassignments are shown in the projections of theROESYspectrumin Figure 5. All four ROESY spectra exhibit the same features: (i)The interiorCDprotonH5 interactswith theBSprotons P21, P22and P23; (ii) the interiorCDprotonsH3 andH30 (theH3next to asubstituted C2) interact with the BS protons P15 and P18; (iii) theproton,Hb, on the hydroxypropyl side chain interacts with theBSproton P19; (iv) no interactions are observed between the exteriorCD protons H2 and H4 and the BS protons. These features aresimilar to the observations made for other CD:BS complexes,37,39,41

and suggest the formation of inclusion complexes in which partsof the BS conjugation tail and D-ring are included in the CDcavity. The interaction between Hb and P19 shows that the HPside chains may interact with the parts of the BS protruding fromthe CD cavity.Isothermal Titration Calorimetry. Figure 6 shows two

examples of the integrated peak area data after subtraction ofthe heat of dilution. From the fit of the model to the data, thecomplexation stoichiometry, n, the molar enthalpy of complexa-tion, ΔH�, and the stability constant, K, were obtained and wereused to calculate ΔG� and ΔS�. The values of K, ΔG�, ΔH�, andΔS� for all complexes are listed in Table 2.Complex Stoichiometries. The complex stoichiometries, n,

yielded as a fitting parameter from themodel fit, in most cases fellwithin the interval 0.9-1.1, suggesting a 1:1 binding mode.However, in some complexes formed with TCDC, n was consis-tently lower than 1, reaching values as low as 0.82 CDs per BS,and titrations involving the highest-substituted CDs (HP102 andHP106) yielded values of n up to 1.2. These deviations from n=1do not change the overall conclusion that they form 1:1 com-plexes, which is in line with previous investigations of CD:BScomplexes,31-37,41 although it has also been reported that TDCand GDC form 2:1 CD:BS complexes with natural βCD30,39,40

and possibly also with modified βCDs.22 The formation of 2:1CD:BS complexes in addition to the formation of 1:1 complexescannot be ruled out, but the association constant for the bindingof the second CD to the BS must be much smaller than for thebinding of the first CD. Otherwise, the chosen model would nothave been able to fit the data and yield stoichiometries close to 1.Stability Constants. The stability constants, K, for the

CD:BS complexes are listed in Table 2 and plotted as a functionof the average degree of substitution in Figure 7. Some of thetrends have previously been observed. In the case of BSs, themostimportant structural feature is the position of hydroxyl groups.The BSs are thought to enter the CD cavity with the side chainfirst, and the presence of a hydroxyl group on C12 stericallyhinders the BS frompenetrating deep into theCD cavity.33,41 As a

Figure 5. Partial ROESYspectrumof the complex ofHP106withGC.Themost important intermolecular interactions betweenCDprotons(H) and BS protons (P) are encircled.

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Figure 6. Titration curves in the case of a strong and a weak complex. The integrated peak area data in panel A is an example of a stronglybinding complex and comes from a titration of 0.253 mMGCDC with 2.50 mMHP063. In the case of a weak complex, the enthalpogramlooks like the one inpanelB,where 1.003mMTCis titratedwith 17.85mMHP106.The fitting parameters corresponding to the best fits (solidlines) are found in the text boxes.

Table 2. Stability Constants, K, Free Energy, ΔG�, Enthalpy, ΔH�, and Entropy, ΔS� of Complexation for All Investigated Complexes

between Bile Salts and HPβCDs of Varying Degrees of Substitution. The Listed Uncertainties Are Calculated as the Standard Deviation

of Three Different Experiments

guest host K (M-1) ΔG� (kJ/mol) ΔH� (kJ/mol) ΔS� (J/mol/K)

TC HP054 1932( 36 -18.76( 0.05 -13.09( 0.30 19.01( 1.16HP063 1512( 71 -18.15( 0.12 -11.73( 0.31 21.51( 1.44HP082 1043( 32 -17.23( 0.07 -6.62( 0.16 35.57( 0.78HP102 861( 48 -16.75( 0.14 -3.50( 0.12 44.44( 0.83HP106 564( 6 -15.70( 0.03 -4.01( 0.08 39.23( 0.36

GC HP054 2141( 87 -19.01( 0.10 -13.56( 0.45 18.28( 1.85HP063 1713( 29 -18.46( 0.04 -12.15( 0.14 21.16( 0.60HP082 1229( 72 -17.63( 0.14 -6.77( 0.38 36.44( 1.69HP102 913( 56 -16.89( 0.16 -3.93( 0.03 43.47( 0.58HP106 669( 38 -16.12( 0.14 -4.28( 0.25 39.71( 1.29

TDC HP054 4052( 174 -20.59( 0.11 -15.56( 0.12 16.87( 0.74HP063 3902( 101 -20.50( 0.06 -13.04( 0.20 25.03( 0.48HP082 3101( 14 -19.93( 0.01 -7.09( 0.03 43.07( 0.14HP102 2632( 21 -19.52( 0.02 -3.82( 0.01 52.68( 0.08HP106 2100( 84 -18.96( 0.10 -3.59( 0.01 51.54( 0.34

GDC HP054 4326( 98 -20.75( 0.06 -16.27( 0.05 15.03( 0.15HP063 4099( 100 -20.62( 0.06 -13.61( 0.11 23.51( 0.58HP082 3284( 72 -20.07( 0.05 -7.76( 0.05 41.30( 0.21HP102 2916( 198 -19.77( 0.17 -4.33( 0.10 51.78( 0.89HP106 2405( 213 -19.29( 0.22 -4.01( 0.13 51.27( 1.16

TCDC HP054 73117( 6446 -27.76( 0.22 -21.88( 0.08 19.70( 1.00HP063 56763( 4257 -27.13( 0.18 -19.49( 0.47 25.62( 1.06HP082 38123( 862 -26.15( 0.06 -15.21( 0.09 36.69( 0.49HP102 28580( 743 -25.43( 0.06 -12.57( 0.08 43.16( 0.38HP106 20027( 102 -24.55( 0.01 -12.02( 0.05 42.05( 0.20

GCDC HP054 84657( 244 -28.13( 0.01 -22.40( 0.03 19.21( 0.10HP063 65768( 919 -27.50( 0.03 -20.36( 0.35 23.96( 1.12HP082 41935( 191 -26.38( 0.01 -16.13( 0.03 34.39( 0.13HP102 30703( 571 -25.61( 0.05 -13.41( 0.03 40.94( 0.26HP106 22617( 226 -24.85( 0.02 -12.83( 0.11 40.33( 0.39

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consequence of no hydroxyl groups at C12, the stability constantsfor the complexes involving the BS anions TCDC andGCDC aremuch higher than for the other BS. Interestingly, the hydroxylgroup on C7 also has a pronounced effect on the complexstabilities. If the 12-OH sterically hinders the BS from a deeperinsertion into the CD cavity, one would not expect the 7-OH toplay a role in complex formation, since this part of the BS is toofar away from the CD. Despite this, the HP-substituted CDs areclearly able to discriminate between BS with and without 7-OH,as seen from the stability constants, which are several times largerwhen 7-OH is missing (TDC and GDC) than when 7-OH ispresent (TC and GC). This suggests that even though the 12-OHsets the limit to how deep the BS are inserted into the CD cavity,the HP-chains on the rim of the CD interacts with the parts of theBS that are outside the CD cavity.

For all investigated complexes, the type of conjugation of theBS side chain only has a minor effect on the complex stabilitiessuch that the glycoconjugated BS form slightly more stablecomplexes than the corresponding tauroconjugated ones. Thisis in accordance with previous results.22,34,41

As seen from Figure 7, the stability constants decrease withincreased DS in a similar way for all types of BS, with theexception that TDC and GDC bind better to the substitutedCDs than to the natural βCD. The decrease in complex stabilitywith increased HP-substitution has previously been observed for

complexation with a number of other guest molecules, includingphenolphthalein19,20,45 and various drug molecules,17 but some-times the complex stability also increases with increasingDS, as isthe case for p-methyl red.19The relation between complex stabilityand DS is discussed in the next section.Thermodynamics of Complexation. The enthalpy of com-

plexation, ΔH�, is negative in all cases, but shows a strongdependence on the DS. As seen in Figure 8, ΔH� increases moreor less linearly with increasingDS resulting in complexations thatare close to being athermal in the case of the highly substituted

Figure 7. Measured stability constants for complexation with TC(9), GC (0), TDC (b), GDC (O), TCDC (2), andGCDC (4) as afunction of the average degree of substitution of the HPβCDs.Stability constants for the natural βCDare taken fromHolm et al.,2009.41

Figure 8. ΔH�, TΔS�, and ΔG� for the complexation of GC (0),GDC (O) and GCDC (4) with HPβCDs of various degrees ofsubstitution. Data for the natural βCDare taken fromHolm et al.,2009.41 Data for the tauroconjugated BS are not plotted, but theyare very similar to the glycoconjugated BS. All data are presentedin Table 2.

(45) Rao., C. T.; Pitha, J.; Lindberg, B.; Lindberg, J.Carbohydr. Res. 1992, 223,99–107.

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Article Sch€onbeck et al.

CDs. This decrease in enthalpic contributions to the complexstabilities is largely compensated by the entropy of complexation,which increases with increasing DS in a similar fashion, as shownin Figure 8. As a result, the free energy of complexation variesrelatively little as a function of the DS (Figure 8). Whereas, ΔH�for the complexation withGC ranges from-24.5 to-4.3 kJ/mol,ΔG� only changes from-19.5 to-16.1 kJ/mol when going fromthe natural β-CD to DS ≈ 1. This significant enthalpy-entropycompensation is illustrated in Figure 9. Enthalpy-entropy com-pensation has previously been observed for a large variety ofCD inclusion complexes14,18,25 including CD:BS complexes.22,41

Though the phenomenonof enthalpy-entropy compensationhasbeen reported a number of times, the molecular (“extra-thermo-dynamic”) mechanism behind this relation is generally highlycontroversial andhas beenargued to bebased uponartifacts.46-52

A discussion of this, however, is beyond the scope of the presentwork.

The effects of side chains substituted onto the rimof theCDhaspreviously been reported to lead to larger ΔH� and ΔS� valuesin the case of the association of larger polycyclic guests withsulfobutylether-βCD18 and for the complex formation betweenmonocarboxylic acid and HPβCD.25 That is in line with theresults presented here. The observations that an increased numberof side chains led to an increase inΔS� were thought to be causedby the additional hydrophobic interactions between the sidechains and the hydrophobic guest molecules.18,25 This interpreta-tion may also explain the variations inΔH� and ΔS� with the DSpresented here.

One might expect that an extended hydrophobic cavity wouldincrease the complex stability, as observed in the case of methyl-ated R- and βCDs,12,53 but the stability constants clearly decreasewith increased substitution. Steric effects arising from the HP-chains partially blocking the cavity entrance could be an impor-

tant factor, but the HP-chains might also alter the rigidity andstructure of theCDand thereby result in a less optimal fit betweenthe CD and the guest molecule. Yong et al.23 have carried outmolecular dynamics simulations of the structural behavior ofnatural βCD and differently substituted HPβCDs in water. Sidechains atO2 induce amore “spreadout” conformationof theCD,where the wide cavity opening is wider and the narrow opening isnarrower. Thismay lead to a less tight fit between the bile salt andthe CD, resulting in lower binding stability, a less favorable ΔH�because of weaker attractive van der Waals interactions, andpossibly a more favorable ΔS� due to a looser fit and increasednumber of conformations of the complex. These thermodynamicconsequences are in accordance with the experimental observa-tions. The simulations also suggest the formation of intramolec-ular hydrogen bonds between the hydroxyl group of the sub-stituent and the hydroxyl group at C3. If these are disrupted uponcomplexation, a less favorableΔH� and a lower complex stabilityis expected.

It is noteworthy that the enthalpy and entropy of complexationchange linearly with the DS, as seen in Figure 8. That means thatthe effects of the HP-chains are additive in the sense that each ofthe added HP-chains contributes equally to the increase in ΔH�.One may view the ΔH� for the complexation with HPβCD asstemming fromΔH� for the complexation with natural βCD plusΔH� for the interaction of each HP-side chain with the bile salt.The effects contributing to ΔS� are additive in the same way.From linear regression of the data for GDC presented in figure 8,onemay conclude that eachHP-substituent adds 3.6 kJ/mol to theenthalpy of complexation and also 3.6 kJ/mol to TΔS� ofcomplexation. In the cases of GC and GCDC, the contributionstoΔH� are 2.8 and 2.2 kJ/mol, respectively, and the contributionsto TΔS� are 2.4 and 1.7 kJ/mol, respectively. If the increase inTΔS� mainly stems from the increased release of ordered watermolecules, the surface area that eachHP-chain dehydratesmay becalculated. Costas et al.54 have calculated that the rearrangementof water molecules near a hydrophobic surface contributes toTΔS�with-38.3mJ perm2 of solute surface area at 298K.Usingthe above increment in TΔS� (2.4 kJ/mol) for each HP chaininteracting with GC yields a dehydrated surface area of 10.4 A2

per HP chain or 2.7 ordered water molecules released forevery HP-chain interacting with GC, using the value of 43.77�10-6 mol of water molecules per m2 of solute from Costas et al.54

This sounds reasonable and supports the interpretation that theincreased release of ordered water molecules is the main con-tributor to the increase in TΔS� with increased DS.

From the enthalpy-entropy compensation plot (Figure 9),it is seen that the complexes can be separated into two clearlydistinguishable groups. All of the complexes involving TCDCor GCDC lie on a straight line in the plot, while the othercomplexes involving the remaining four bile salts form a secondgroup. This second group might be separated into two groups:one group for the TDC andGDCbile salts and another group forthe TC and GC bile salts. The linear correlation of TΔS� withΔH�within a series of complexes suggests that the overall bindingmode is the same for all the complexes in that series. Thus, thebinding mode of TCDC and GCDC to HPβCD is different thanthe binding mode of TDC/GDC/TC/GC to HPβCD. This con-clusion is in line with the observation that TCDC and GCDCpenetrate deeper into the CD cavity than the other bile salts.Interestingly, the number of HP-chains does not change theoverall binding mode. It is the structure of the bile salt molecule

Figure 9. Entropy-enthalpy compensation plot for all investi-gated complexes. GC and TC (0); TDC and GDC (O); TCDCand GCDC (4). Data for the natural βCD (lower left corner) aretaken from Holm et al., 2009.41

(46) Liu, L.; Guo, Q.-X. J. Incl. Phenom. Macrocycl. Chem. 2002, 42, 1–14.(47) Liu, L.; Guo, Q.-X. Chin. J. Chem. 2001, 19, 670–674.(48) Exner, O. Prog. Phys. Org. Chem. 1973, 10, 411–482.(49) Wold, S.; Exner, O. Chem. Scr. 1973, 3, 5–11.(50) Exner, O. Nature 1964, 201, 488–490.(51) Krug, R. R.; Hunter, W. G.; Grieger, R. A. J. Phys. Chem. 1976, 80, 2335–

2341.(52) Krug, R. R.; Hunter, W. G.; Grieger, R. A. J. Phys. Chem. 1976, 80, 2341–

2351.(53) Castronuovo, G.; Niccoli, M. J. Incl. Phenom. Macrocycl. Chem. 2007, 58,

289–294.(54) Costas,M.; Kronberg, B.; Silveston, R. J. Chem. Soc., Faraday Trans. 1994,

90, 1513–1522.

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rather than the number of HP chains that determines the overallbinding mode.

The slope (R) and intercept (TΔS0) of the enthalpy-entropycompensation plot has previously been interpreted asmeasures ofthe degree of conformational changes and the degree of desolva-tion, respectively.14 A slope close to 1 means that the complexpossesses considerable flexibility and can undergo significantconformational changes upon complexation, whereas the inter-cept reflects the entropic contribution to complex stability whenthere is no enthalpic contribution and is related to the degree ofdesolvation of the host and guest molecules upon complexation.From the enthalpy-entropy compensation plot, it is evident thatthe intercept for the TCDC/GCDC bile salts (TΔS0≈ 22 kJ/mol)is larger than for the other bile salts (TΔS0 ≈ 18 kJ/mol). Again,this is consistent with the deeper insertion of the TCDC/GCDCbile salts into the CD cavity and the associated larger extent ofdehydration. For comparison, the analysis of more than 1000different complexes with natural CDs and more than 100 mod-ified CDs yielded intercepts of 12 and 17 kJ/mol, respectively.14 Itis clear that the interaction between HPβCD and TCDC/GCDCbile salts results in an unusually large value ofTΔS0 suggesting thatdehydration plays an exceptionally large role in these complexes.

Conclusion

Stability constants and the associated enthalpies and entro-pies of complexation were determined by isothermal titration

calorimetry for complexes between six bile salts and hydroxypropy-lated β-cyclodextrins with varying degrees of substitution. Theenthalpy of complexation increases strongly and linearly with thedegree of substitution, but this is largely compensated by a similarincrease in the entropy of complexation, so that the standard freeenergy of complexation increases slightly with the degree ofcyclodextrin substitution.

The overall complex structure and stability is determined by thestructure of the bile salt and in particular the presence or absenceof the C-12 hydroxyl group, as previously described for naturalβ-cyclodextrin.30,41 The hydroxypropyl chains on the rim of thecyclodextrin are thought to align themselves with the hydro-phobic parts of the bile salt molecule that protrudes from thecyclodextrin cavity. This results in a release of ordered watermolecules, which is responsible for the significant variations in theenthalpy and entropy of complexation, but has a somewhatsmaller effect on the complex stability. It is estimated that eachhydroxypropyl chain dehydrates a surface area of around 10 A2.

Since the hydroxypropyl chains have the same qualitativeeffect on the complexation with all six different bile salts, onemay expect a similar effect on complexation with other guestmolecules.

Supporting Information Available: Mass spectra andselected 1D and 2D NMR spectra of the CDs. This materialis available free of charge via the Internet at http://pubs.acs.org.