Published: April 21, 2011

r 2011 American Chemical Society 5832 dx.doi.org/10.1021/la200381f | Langmuir 2011, 27, 5832–5841

ARTICLE

pubs.acs.org/Langmuir

Methylated β-Cyclodextrins: Influence of Degree and Pattern ofSubstitution on the Thermodynamics of Complexation withTauro- and Glyco-Conjugated Bile SaltsChristian Sch€onbeck,†,‡ Peter Westh,† Jens Christian Madsen,§ Kim Lambertsen Larsen,||

Lars Wagner St€ade,|| 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

)Section of Chemistry, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University,Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark

bS Supporting Information

ABSTRACT:

The complexation of 6 bile salts with various methylated β-cyclodextrins was studied to elucidate how the degree and pattern ofsubstitution affects the binding. The structures of the CDs were determined by mass spectrometry and NMR techniques, and thestructures of the inclusion complexes were characterized from the complexation-induced shifts of 13C nuclei as well as by 2DROESYNMR. Thermodynamic data were generated using isothermal titration calorimetry. The structure�properties analysis showed thatmethylation at O3 hinders complexation by partially blocking the cavity entrance, while methyl groups at O2 promote complexationby extending the hydrophobic cavity. Like in the case of 2-hydroxypropylated cyclodextrins, the methyl substituents cause anincreased release of ordered water from the hydration shell of the bile salts, resulting in a strong increase in both the enthalpy and theentropy of complexation with increased number of methyl substituents. Due to enthalpy�entropy compensation the effect on thestability constant is relatively limited. However, when all hydroxyl groups are methylated, the rigid structure of the free cyclodextrinis lost and the complexes are severely destabilized due to very unfavorable entropies.

’ INTRODUCTION

Cyclodextrins (CDs) are ring-shaped molecules often describedas truncated cones. Due to their hydrophilic outer surface andhydrophobic inner cavity they are able to form inclusion complexeswith a large variety of predominantly hydrophobic guestmolecules.1,2 This makes CDs useful for many applications,3 espe-cially within pharmaceutical4 and food sciences. At present thereexist several marketed pharmaceutical products containing CDs.5,6

The most common cyclodextrins are R-, β-, and γCDs,differing in the number of glycopyranose units and thereforediameter of the cavity, which increases in size from R to γ. Theypresent 18, 21, and 24 modifiable hydroxyl groups, respectively,distributed with 2/3 on one rim (often termed the wide rim) and1/3 on the other (termed the narrow rim). Great efforts have

been put into developing modified CDs with altered propertiescompared to the native CDs and in controlling the degree andpattern of the substitution. Hence, a large number of modifiedCDs exist in which various substituents are attached to theoxygens in the 2-, 3- and 6-positions (see Figure 1). Introductionof substituents alters both the physicochemical properties of theCDs and the ability to form complexes.7�13 Thus, if the effects ofthe different substituents are well understood, one might tailorCDs for specific guest molecules and for specific purposes inorder to achieve optimal properties.

Received: January 28, 2011Revised: April 5, 2011

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The primary driving forces for complexation with CDs arebelieved to arise from hydrophobic and van der Waals interactions,with contributions from other forces such as hydrogen bonds, ifpresent.2 Recently, a systematic study of 2-hydroxypropylatedβCDs (HPβCDs) was published in which the effects of the degreeof substitution (DS) on the thermodynamics of complexation withbile salts (BSs) was investigated.12 Although the DS had only aminor influence on the association constants, it had a stronginfluence on the enthalpy and entropy of complexation, bothincreasing linearly with increasing DS. This was interpreted asthe result of an increased release of ordered water from thehydration shells of the guest molecules and the hydroxypropylsubstituents. In addition to HPβCD, methylated CDs are amongthe most commonly used CDs. Therefore, a systematic investiga-tion of how the degree and pattern of substitution affect the abilityof methylated βCD (mβCD) to form inclusion complexes willprovide useful information for the utilization of this class of CDs.

Furthermore, an investigation of the effects of the degree andpattern of methylation on the thermodynamic parameters, ΔH�and ΔS�, associated with complexation will reveal some of theeffects driving the interaction.

There are several structural differences between the methyland HP substituents that might affect the complexation proper-ties of the CD: (1) The HP chain contains a hydroxyl group thatis expected to interact with the surrounding water molecules andmay also participate in intramolecular hydrogen bonds, assuggested by molecular dynamics simulations,14 (2) the HPchain is more flexible, and (3) the HP chain has a larger surfacearea andmay therefore interact with and dehydrate larger parts ofthe hydrophobic guest molecule. Another interesting aspect isthe commercial availability of mβCDs spanning a larger range ofDS. The largest DS of the investigated HPβCDs was 1.06 (onaverage 1.06 2-hydroxypropyl chains per glycopyranose unit),but both dimethylated (DS = 2) and permethylated (DS = 3)βCDs are available. The predominant site of substitution on theHPβCDs was O2, but the larger DS of the mβCD forces asignificant fraction of the substituents to be located at the twoother sites on the glycopyranose units, i.e., O3 and O6. It was notpossible to precisely determine the substitution pattern of theHPβCDs, but in the present work the substitution patterns of themβCDs are determined, and this enables a discussion of theeffects of having substituents at the various sites of the CD. Thismay provide useful information about the thermodynamic im-portance of the site of substitution.

A large number of studies investigating the complexes ofmβCDswith various guestmolecules are published in the literature.The majority of these studies investigate dimethylated or trimethy-lated CDs where two or all of the substitution sites (O2, O3, andO6) are completely methylated. Only a small number of studiesfocus on randomly methylated βCDs (RAMEB) where each of the

3 sites is only partially methylated. This lack of attention might bedue to the fact that all RAMEB samples are mixtures of manyisomers and that the samples need to be characterized with respectto the degree and pattern of substitution before a detailed analysisand discussion concerning the effects of the substituents can bemade. However, RAMEBs are widely used as pharmaceuticalsolubilizers,5 and it is therefore important to understand thestructural factors governing the behavior of this class of mβCDs.Also, from an academic point of viewRAMEBs are interesting sincepartial methylation allows the intramolecular hydrogen-bondnetwork15 to be partially retained. Thus, it might be possible toobserve the steric and hydrophobic effects of the methyl groupswhile avoiding the effects of the complete elimination of thehydrogen-bond network that occurs when O2 and O3 are com-pletely methylated. In the present work both dimethylated andtrimethylated βCDs as well as a range of RAMEBs are studied, andall CD samples are thoroughly characterized with respect to theaverage number and position of substituents and the distribution inthe number of substituents within each sample. To our knowledge,such a comprehensive study of the complex forming capabilities ofwell-characterized RAMEBs has not previously been conducted.

In order to clarify the effects of methyl substituents, thepresent study investigates the complexing abilities of methylatedβCDs having a wide range of DS by use of isothermal titrationcalorimetry (ITC). This technique provides precise values ofassociation constants, K, as well as direct determination ofenthalpies (ΔH�) and entropies (ΔS�) of complexation andthereby provides useful thermodynamic insight into CD chem-istry. The guest molecules in the present study are 6 different bilesalts (see Figure 2) present in the intestine of man, rat, and dog.16

They are of interest as they have a strong affinity for βCD17�23

and γCD24 and because the interaction with BS affects therelease profile following oral administration of a compound in aCD complex.4,25 In order to assist a structural interpretation ofthe thermodynamic parameters, the complexes are structurallycharacterized by ROESY-NMR and the complexation-inducedshifts (CIS) of 1H and 13C NMR peaks.

’EXPERIMENTAL SECTION

Materials. Sodium salts of the bile acids taurocholate (TC),taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), glyco-cholate (GC), glycodeoxycholate (GDC), and glycochenodeoxycholate(GCDC) as well as natural βCD were purchased from Sigma-Aldrich(St. Louis, MO). Samples of randomly methylated βCD having nominalaverage DS of 0.6, 1.2, and 1.8 were acquired from Wacker Chemie(Burghausen, Germany), and a sample with a nominal DS of 0.57 was agenerous gift from Roquette (Le Strem, France). A single sample ofrandomly methylated βCD with no specification of DS was purchasedfrom Sigma-Aldrich along with two samples of heptakis(2,6-di-O-methyl)-βCD and a single sample of heptakis(2,3,6-tri-O-methyl)-βCD. In the following, the CD samples will be named according tothe DS determined experimentally by mass spectrometry. The names, aswell as the degree and pattern of substitution, appear fromTable 1.Milli-Q water was used for all solutions, and all chemicals were of analyticalgrade or higher and used without further purification.Mass Spectrometry. MALDI-TOF MS was performed using a

matrix consisting of fast evaporating nitro-cellulose and R-cyano-4-hydroxycinnamic acid (CCA) on a Reflex III (Bruker Daltonics, Bre-men, Germany). 10 mM solutions of the mβCD samples dissolved in a1:4 (v/v) mixture of nitro-cellulose and saturated CCA in an aqueoussolution of 0.1% trifluoroacetic acid and 80% acetonitrile were applieddirectly to the MALDI target plate.

Figure 1. Numbering of the nuclei in methylated βCD.

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NMR Spectroscopy. For characterization of the CD samples,standard 1H spectra, attached proton test (APT), and two-dimensionalHSQC,26 COSY, and HMBC27 spectra were recorded at 25 �C on aBruker Avance-600 NMR spectrometer operating at 14.1 T andequipped with a cryogenically cooled probe. The experiments wereperformed on 10 mMCD samples in D2O. For the structural character-ization of the complexes, 2DROESY28 spectra were recorded in additionto the above-mentioned experiments. The concentrations of CD and BSin the D2O solutions were both 10 mM. For the assignment of the freeBS anions GC, GDC and GCDC, H2BC29 spectra were recorded inaddition to APT, HSQC, and HMBC spectra. To avoid micellization ofthe BSs the concentrations were 2 mM in the assignment samples.Isothermal Titration Calorimetry (ITC). Solutions were made

by weighing out the dried powders of CD and BS and dissolving them in50mMphosphate buffer pH 7.1. The reaction cell of the calorimeter wasfilled with BS solutions ranging from 0.25 to 1 mM and was titrated withCD solutions in the concentration range 2.5�20 mM. The higher thestability constants, the more diluted the solutions. Concentrations of theCD solutions were calculated from the average molecular weight of eachCD sample as determined by mass spectrometry. The titrations wereperformed in triplicate on aMicrocal VP-ITC titration microcalorimeter(MicroCal, Northhampton, MA) at 25.0 �C and atmospheric pressure.

Heats of dilution were measured in separate runs by titrating the CDsolutions into buffer solution. The measured heats of dilution weresubtracted from the titration heats to yield the heats of complexation.

The titration curves were fitted using MicroCal’s ITC data analysisapplication to the Origin software package (version 7.0) assuming asingle set of identical and independent binding sites. This yields thecomplexation stoichiometry (n), the molar enthalpy of complexation(ΔH�), and the stability constant (K). The stability constant and molarenthalpy enables calculation of the standard Gibbs free energy ofcomplexation (ΔG�) and the standard change in entropy (ΔS�)according to

ΔG� ¼ � RT ln K ¼ ΔH�� TΔS�

where R is the gas constant and T is the temperature in Kelvin.

’RESULTS AND DISCUSSION

Characterization of Cyclodextrin Samples. MALDI-TOFmass spectrometry revealed that only the m300 sample wasmonodisperse. All other samples consisted of a mixture of CDshaving different DS. The randomly methylated CD samples were

Table 1. Average Degree of Substitution and Distribution of Substituents

CD reported DS from supplier measured DS (MS) measured DS (1H NMR) DS(O2)a DS(O2)b DS(O3)b DS(O6)b

m067 0.6 0.67 0.44 0.36 0.38 0.19 0.10

m069 0.57 0.69 0.63 0.52 0.52 0.12 0.05

m117 1.2 1.17 1.03 0.55 0.59 0.33 0.25

m163 1.63 1.28 0.65 0.62 0.34 0.67

m167 1.8 1.67 1.50 0.64 0.63 0.37 0.67

m209 2 2.09 1.90 1.00 1.00c 0.09c 1.00c

m212 2.16 2.12 1.95 1.00 1.00c 0.12c 1.00c

m300 3 3.00 2.99 1.00 1.00d 1.00d 1.00d

aDS(O2) is determined from peak areas of H1 and H10. bDetermined from peak areas of the methyl carbons in combination with DS fromMS. cThereare no signs of unsubstituted O2 or O6. Therefore, it is assumed that these sites are fully substituted and that the remaining methyl groups are situated atO3. dAll spectra show that the sample consists of heptakis(2,3,6-tri-O-methyl)-βCD.

Figure 2. Names and structures of the 6 investigated bile salt anions.

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the most polydisperse with 6�9 CDs of different masses with anormal distribution around the average molecular mass. Thelargest peaks in the mass spectra of m209 and m212 corre-sponded to heptakis(2,6-di-O-methyl)-βCD, but significantamounts of higher substituted CDs were also present. All massspectra are found in the Supporting Information.From the peak intensities in the mass spectra the average DS

were calculated as

DS ¼∑iIi 3DSi

∑iIi

where Ii denotes the intensity of the ith peak andDSi denotes thedegree of substitution corresponding to the ith peak. Thecalculated DS for the investigated CDs are found in Table 1

along with the nominal DS reported by the supplier. In general,there was a good correlation between the nominal DS and thefound one.The distribution ofmasses and the averageDSmaybe determined

byMS, but the pattern of substitutionmust be determined byNMR.From analysis of 1D 1H NMR, 1D 13C NMR, and 2D HSQC andHMBC spectra, the peaks in the 1D spectra were assigned as shownin Figures 3 and 4 and the Supporting Information. In agreementwith previous observations,13,30 the directly substituted carbons (C2,C3, and C6) were shifted ∼10 ppm downfield relative to theirunsubstituted counterpartswhile theneighboring carbons experienceupfield shifts between 1 and 3 ppm. Protons on directly substitutedcarbons are shifted upfield by 0.16�0.27 ppm, while protons onneighboring carbons are shifted downfield by 0.06�0.19 ppm.Theseshifts are observed in all of themethylated CDswith the exception ofm300,where the chemical shifts ofmany peaks differ somewhat from

Figure 3. Partial overlaid HSQC (black) and HMBC (red) spectra of m167. The assignment of the 1H and 13C peaks are shown in the horizontal andvertical projection, respectively. The subscript Nsub denotes the directly substituted carbon (or the proton attached to this carbon), while theneighboring carbon (or its proton) is denoted by a prime N0.

Figure 4. 1H NMR spectrum of m067. The assignment was made from the HSQC and HMBC spectra.

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the corresponding peaks in the other CDs. This suggests a distortionof the CD ring induced by disruption of intramolecular hydrogenbonds. Judging from the chemical shifts, this distortion does nothappen gradually as the DS increases but only when all hydroxylgroups are substituted.The average degree of substitution may be quantified from the

proton peak areas. As seen on Figure 4, the only peaks that arenot overlapping are H1 and H10, so the DS can be determined bycomparing the sum of these two peaks (which integrates 1proton) to the summed peak areas of all other protons. Theresults are listed in Table 1. The DS obtained from 1H NMR areconsistently lower than the ones obtained from MS. From themass spectra it is clear that the DS of m209 and m212 must belarger than 2, and since the 1H NMR yields values lower than 2,the DS determined by MS are assumed to be the most reliable.Not only the total DS may be quantified by 1H NMR but alsothe degree of substitution on O2 [DS(O2)] may be quantifiedfrom the relative areas of H1 and H10 and are also shown inTable 1.The use of 13C peak areas for quantification is usually associated

with some errors due to different NOEs and T1 relaxation times,but as Tongiani et al.30 argued, the areas of the methyl groups maybe compared to each other and the area of a substituted carbonmay be compared to the corresponding unsubstituted carbon.Thus, the degree of substitution on each site may be calculatedfrom the areas of substituted and unsubstituted carbon peaks.However, due to partially overlapping peaks, this is not possible inall cases. A better method may be to calculate the relativedistribution of substituents by comparing the areas of the methylcarbons. Once the fraction of the methyl groups attached to C2 isknown, DS(O2) may be calculated by multiplying with the totalDS. DS(O3) and DS(O6) are calculated in the same way and arelisted in Table 1. From these results it is apparent that a wide rangeof the number and positions of substituents is evaluated in thepresent study.

Structural Characterization of Complexes.Amore qualifiedinterpretation of the measured thermodynamic parameters canbe obtained if the structures of the complexes are established. Forstructural characterization of the complexes, two different NMRmethods were employed. The first is 2D ROESY in whichprotons that are spatially close give rise to off-diagonal cross-peaks whose intensities depend on the distance between theprotons. The cross-peaks between CD protons and BS protonswill thus reveal the orientation of the BS inside the CD.The second method exploits the fact that the chemical shifts of

NMR-active nuclei depend on their chemical environments.When the water molecules surrounding the free BS moleculeare replaced by the hydrophobic interior of the CD, the peaks ofthe BS nuclei experiencing this change in environment will movein the NMR spectrum. This complexation-induced shift (CIS)can be determined for 1H and 13C nuclei and has previously beenused to determine the inclusion site of the guest molecule.13,31�34

These two methods were used to investigate the structures ofthe 12 complexes formed between the 3 BSs GC, GDC, andGCDC and the 4 CDs βCD, m167, m209, and m300.The assignment of the 13C and 1H nuclei of the free BS anions

are found in the Supporting Information. The assignments are inagreementwith the literature13,35,36 and are shown together with atable of all 13CCIS and selected ROESY spectra in the SupportingInformation.Complexes with GCDC. From both the ROESY spectra and

the CIS of the BS 13C it is clear that all 4 investigated CDs formsimilar 1:1 inclusion complexes with GCDC. The CDs encapsu-late parts of the C and D rings of the steroid structure and part ofthe conjugated side chain of GCDC, as previously reported.13,21

It is apparent that the GCDCnuclei 12, 13, 16, 17, 18, and 20�24all have large CIS, strongly suggesting that these nuclei areincluded within the CDs. Interestingly, nuclei 11 and 15 exhibitlarge CIS in the complex with m300 but not in the complexeswith βCD andm209. This suggests that the methyl substituent atO3, which is present in m300 but not in βCD and m209, extendsthe hydrophobic cavity to also include nuclei 11 and 15. Thepolydisperse m167, which is partially substituted at O3, alsoinduces a large CIS on nucleus 15 but not on nucleus 11.The most important features of the ROESY spectra are the

interactions between the interior CD protons H3 andH5 and theBS protons (denoted with a P to distinguish them from the CDprotons). H3 is located close to the wide opening of the CD,while H5 is located closer to the narrow opening. The strongROESY interactions between H3 and P18 and between H5 andP21 suggest that GCDC enters the CDs from the wide opening,as previously observed for the natural βCD.21 In addition, H5interacts with protons on the side chain and the D ring, while H3interacts with protons on the C and D rings. From theseobservations and the 13C CIS described above, the complexstructure shown in Figure 5A is deduced for the complexesbetween GCDC and the 4 investigated CDs.Complexes with GC.The APT spectra of GC complexes suffer

from a large broadening of many peaks, and several peakscompletely disappear in the noise. It is apparent that it is thenuclei at the C and D rings and the side chain that are broadened,while the nuclei at the A and B rings are much less affected. Sincethe broadened nuclei are also the ones that experience the largest13C CIS, it may be concluded that the broadening is due toinclusion of GC in the CD and that the structure of the complexis quite similar to the ones formed with GCDC. The structureis supported by the ROESY spectra and is consistent with

Figure 5. (A) Suggested structure of the 1:1 complexes betweenGCDC and all CDs. (B) Suggested structure of the 2:1 CD:BScomplexes between GDC and all CDs except m300. The cyclodextrinsare represented by their cavities whose dimensions more or lesscorrespond to the ones given by Szejtli.46

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previously proposed structures of similar CD:BS complexes.13,20,21

However, the situation might be different for the complex GC/m300 in which the peak broadening described above occurs, butthe 13C CIS are all small, and no correlation peaks between H3and H5 and the GC protons are observed in the ROESYspectrum. This indicates a very limited or no inclusion of GCinto m300.Complexes with GDC. Peak broadening was also observed in

the APT spectra of the GDC complexes. In contrast to the GCcomplexes, not only are the nuclei at the C and D rings and theside chain broadened but also most of the nuclei at the A and Brings are broadened. The nuclei least affected by broadening arethose in the middle of the BS, e.g., numbers 8, 9, and 10. Almostall nuclei have relatively large CIS, and together with the peakbroadening pattern, this suggests that the CDs bind to both endsof the BS forming 2 types of complexes or to some extent 2:1 CD:BS complexes as depicted in Figure 5B. The binding of the CDsto the A and B rings of the BS is confirmed by the ROESY spectra,in which there are many correlation peaks between the interiorCD protons H3 and H5 and several protons on the A and B ringsof GDC. H3 tends to interact strongest with P4 and P6, while H5interacts strongest with P2 and P4, thereby confirming thestructure in Figure 5B, in which the BS enters both CDs throughthe wide openings. Again, the complex GDC/m300 might be anexception since the ROESY cross-peaks are few, weak, and hardto assign. It is clear, however, that an inclusion complex is formedsince ROESY interactions are observed between the interior CDprotons H3 and H5 and a number of BS protons, including astrong interaction between H5 and P21 and a weak interactionbetween H3 and P19. Interestingly, in contrast to all of the otherinclusion complexes, no interaction between H3 and P18 isobserved.The binding to a second binding site or formation of 2:1 CD:

BS complexes has previously been observed for complexesbetween natural βCD and this type of BS that has no hydroxylgroup on C7 and therefore permits inclusion of the A and Brings.17,20,37 The present investigation shows that all mβCDs,with the possible exception of m300, behave similarly.Conformation of CDs in Complexes. X-ray diffraction studies

of crystalline complexes of permethylated CDs have shown thatthe methyl groups on O2 (2-CH3) of the permethylated CDspoint away from the center of the CD cavity, while the 3-CH3

groups point toward the center of the cavity opening.38 Thepresent study strongly suggests that this orientation of themethylgroups is maintained in the complexes in aqueous solution:numerous ROESY correlation peaks are seen between 3-CH3

and various BS protons, whereas 2-CH3 only interacts with P19or no BS protons at all. Furthermore, large 13C CIS are seen for3-CH3 in the complexes of m300 with GDC and GCDC, but theother two methyl substituents exhibit relatively small CIS.Among the 13C nuclei on the CD, it is C1 and C4 that

experience the largest CIS (Supporting Information) whencomplexed with one of the BS. The CIS of these two nuclei varygreatly between the CDs and increase in the order βCD < m209< m167 , m300, with the CIS in m300 reaching 3.26 and 4.81ppm for C1 and C4, respectively. The C1 and C4 carbonsparticipate in the glycosidic bonds between the methylglucoseresidues, and the large CIS of these nuclei probably reflects largeconformational changes upon complexation, in which the tiltangles of the methylglucose residues are significantly changed.The flexibility of the CDs increases with increased substitutiondue to disruption of the H-bond network between the secondary

hydroxyl groups, and this explains why the CIS of C1 and C4increases in the observed order.39 Apparently, m300 undergoeslarge conformational changes upon complexation, whereas thenatural βCDhas a muchmore rigid structure. Interestingly, theselarge CIS are not observed for complexation of m300 with GC,adding to the assumption that these two molecules do not forman inclusion complex.The observed CIS for complexation of GCDC with βCD and

m300 are quite similar to those observed for complexation of neutraland charged porphyrins with the same two CDs.40,41 Despite thedifferent characters of the guestmolecules they induce similarCIS onthe 13C nuclei of the CDs, and this strongly suggests that it is theconformational changes of the CDs that are responsible for the CIS.Isothermal Titration Calorimetry. Calorimetric titrations

were performed in triplicate for all combinations of the 9 CDsand 6 BS. Each CD sample, with the exception of the naturalβCD andm300, is actually a mixture of many differentmβCDs ofvarious masses and substitution patterns. This means that eachenthalpogram represents not just a single complexation equilib-rium but many competing equilibria, each characterized by anindividual equilibrium constant. An exact model of such systemconsisting of many linked equilibria would be too complex toprovide meaningful fits to the enthalpograms. However, asevidenced by the mass spectra there is a relatively narrowdistribution in the number of substituents per CD within eachsample. Therefore, it might be assumed that all CDs within asample have somewhat similar affinities toward the BS. In suchcase the multiple binding equilibria will essentially behave like asingle equilibrium describing the complexation between the BSand a single “average”mβCD characterized by the average DS ofthe sample. On the basis of this assumption the enthalpogramswere fitted with a model based on one set of identical andindependent binding sites. This model provided good fits to allenthalpograms and yielded stoichiometries of about 1, therebyvalidating the above assumption. Representative enthalpogramsare shown in the Supporting Information.Although the NMR study of the complex structures provides

evidence of a secondary binding site on the GDC anions, thissecondary interaction is apparently too weak to be detected bythe method used in the ITC studies. If ΔG� and ΔH� for thesecondary binding site are small compared to the primary site, theheats produced by binding to the secondary site are so small thatthey “vanish” in themuch larger heats produced by binding to theprimary site.The titrations of GC/TC and GDC/TDC with m300 all

produced very small heats. Compared to these small heats theenthalpy of dilution of m300 is relatively large and the subtrac-tion of the enthalpy of dilution therefore introduces relativelylarge errors on the corrected reaction enthalpies. Although theone set of sites model fits the reaction enthalpies pretty well theenthalpograms show no real signs of saturation and it isconcluded that the enthalpies of complexation and/or thestability constants are too small to be correctly determined byITC. Titrations of GC and TC with m300 conducted by Liuet al.42 yielded very small stability constants on the order of200 M�1. In accordance with the discussions in the NMRsection, it may be concluded that GDC/TDC and GC/TC formweak inclusion complexes with m300.Thermodynamics of Complexation. In addition to the stoi-

chiometries, the fitting of the model to the enthalpograms yieldedvalues for the stability constant (K) and the molar enthalpy ofcomplexation (ΔH�), from which the standard Gibbs free energy

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(ΔG�) and the molar entropy of complexation (ΔS�) can becalculated. These are shown in Table 2, and representative fits areshown in the Supporting Information. The relationship betweenthe BS structures and the stabilities of complexes formedwith βCDand modified βCDs has previously been described.13,17,18,21,43,44

The lack of a hydroxyl group on C12 allows a deeper inclusion ofGCDC and TCDC, and consequently, the stability constants aremuch larger than for the other BSs, which possess a hydroxyl groupat C12. The presence of a hydroxyl group at C7 has an analogousbut less pronounced influence on the stability constants as seen bythe larger stability constants of complexes with GDC/TDCcompared to complexes with GC/TC. The type of amino acidconjugation only has aminor influence in that the glyco-conjugatedBSs have slightly larger stability constants than the tauro-conjugatedones. These general trends are also seen for the methylated βCDsin the present study with the small exception that m209 and m212form more stable complexes with TC than with GC.The influence of the DS of the mβCDs on the measured

stability constants of the complexes is shown in Figure 6. Unlikethe HPβCDs,12 in which the stability constant systematicallydecreased with increasing DS, no simple relation between thestability constant and the DS was seen for the investigatedmβCDs. However, binding to all types of BSs is similarly affectedby the degree of substitution. Small DS (∼0.7) increases the

binding to all BSs relative to the natural βCD, but furthermethylation decreases the affinity with a local minimum atDS ≈ 1.7. Then the stability constants increase for m209 andm212 and decrease significantly in the case of m300, which hasvery poor binding properties compared to the other CDs. Toexplain these observations it is necessary to take into account theposition of the methyl substituents instead of just considering thetotal number of substituents. A methyl substituent affects thecomplexation in different ways depending on whether it issituated at O2, O3, or O6. The substitution patterns of themβCDs used in the present study are presented in Table 1. Theobtained stability constants suggest that methylation at O3severely hinders complexation while methylation at O2 improvescomplexation. At low DS (∼0.7), most of the methyl groups aresituated at O2 and this improves the binding relative to thenatural βCD. This increase is more pronounced for m069 thanfor m067, both having DS ≈ 0.7, since the former has moremethyl groups at O2 and fewer at O3. At higher DS, the increasein the number of methyl groups mainly takes place at O3 and O6and this causes the stability constants to decrease. However,m209 and m212 are both completely methylated at O2 and onlyslightly methylated at O3, and therefore, they bind more stronglyto the BSs than m163 and m167, in which more of thesubstituents are at O3. m212 has a slightly higher DS than

Table 2. Stability Constants, K, Free Energy, ΔG�, Enthalpy, ΔH�, and Entropy, ΔS� of Complexation for All InvestigatedComplexes between Glyco-Conjugated Bile Salts and mβCDs of Varying Degrees of Substitutiona

guest host K (M�1) ΔG� (kJ/mol) ΔH� (kJ/mol) ΔS� (J/mol 3K)

GC βCD 2876 ( 81 �19.7 ( 0.1 �25.7 ( 0.2 �19.9 ( 0.9

m069 3464 ( 77 �20.2 ( 0.1 �20.0 ( 0.2 0.9 ( 1.0

m067 2902 ( 51 �19.8 ( 0.0 �19.2 ( 0.4 1.9 ( 1.1

m117 2262 ( 78 �19.2 ( 0.1 �13.7 ( 0.2 18.1 ( 0.9

m163 1809 ( 39 �18.6 ( 0.1 �12.4 ( 0.2 20.9 ( 0.9

m167 1742 ( 18 �18.5 ( 0.0 �12.5 ( 0.1 20.3 ( 0.2

m212 2474 ( 26 �19.4 ( 0.0 �16.0 ( 0.1 11.4 ( 0.5

m209 2716 ( 257 �19.6 ( 0.2 �15.1 ( 0.7 15.0 ( 3.2

m300 NA b NA b NA b NA b

GDC βCD 3075 ( 71 �19.9 ( 0.1 �31.3 ( 0.2 �38.2 ( 0.8

m069 7374 ( 111 �22.1 ( 0.0 �22.3 ( 0.0 �0.8 ( 0.2

m067 5321 ( 80 �21.3 ( 0.0 �21.0 ( 0.1 1.0 ( 0.5

m117 4825 ( 25 �21.0 ( 0.0 �12.9 ( 0.1 27.1 ( 0.3

m163 4265 ( 242 �20.7 ( 0.1 �10.6 ( 0.2 33.9 ( 1.3

m167 4207 ( 213 �20.7 ( 0.1 �10.6 ( 0.3 33.7 ( 1.2

m212 7514 ( 91 �22.1 ( 0.0 �20.4 ( 0.2 5.9 ( 0.8

m209 8365 ( 98 �22.4 ( 0.0 �19.8 ( 0.2 8.6 ( 0.7

m300 NA b NA b NA b NA b

GCDC βCD 139 733 ( 4852 �29.4 ( 0.1 �31.0 ( 0.2 �5.4 ( 0.4

m069 191 200 ( 1652 �30.2 ( 0.0 �28.6 ( 0.1 5.1 ( 0.3

m067 179 000 ( 5981 �30.00 ( 0.1 �28.1 ( 0.6 6.2 ( 1.7

m117 152 767 ( 3493 �29.6 ( 0.1 �23.0 ( 0.1 22.0 ( 0.1

m163 123 967 ( 2023 �29.1 ( 0.0 �22.2 ( 0.3 23.4 ( 0.8

m167 124 267 ( 839 �29.1 ( 0.0 �21.8 ( 0.2 24.4 ( 0.6

m212 141 100 ( 2689 �29.4 ( 0.1 �27.7 ( 0.0 5.5 ( 0.2

m209 151 833 ( 3516 �29.6 ( 0.1 �27.0 ( 1.0 8.8 ( 3.2

m300 2671 ( 350 �19.5 ( 0.3 �34.8 ( 2.4 �49.2 ( 7.8aA similar table containing the thermodynamic parameters for the tauro-conjugated bile salts is found in the Supporting Information. The listeduncertainties are calculated as the standard deviation of 3 different experiments. bThe association between the CD and the BS is too weak to be reliablydetermined by ITC.

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m209, and since the O2 and O6 sites are probably completelysubstituted, the extra methyl groups on m212 must be situated atO3. This agrees well with its lower binding properties. Theadditional full methylation at O3 dramatically reduces thecomplexation properties of m300. A comparison of m117 withm167 and m169 indicates that methylation at O6 reduces thestability constant. However, the negative influence of methyla-tion at O6 seems to be relatively weak and does not significantlydisturb the good binding properties of m209 and m212 resultingfrom the large number of substituents at O2 and the smallnumber at O3.As discussed in the NMR section, the methyl groups at O3

seem to point toward the center of the cavity opening and it istempting to suggest that these methyl groups hinder inclusion bypartially blocking the cavity entrance.10 Methyl groups at O2,which point away from the entrance, do not hinder inclusion andmay favor inclusion by extending the hydrophobic cavity of theCDs.10 The low affinity of m300 toward GC/TC and GDC/TDC could very well be caused by unfavorable interactionsbetween the hydroxyl group on C12 on the BS and the methylgroups on O3 of m300. As discussed above, the hydroxyl groupon C12 of the BS hinders a deep inclusion of the BS and causes a

strong decrease in the stability constant. The presence of methylgroups on all O3s and their interactions with the C12 hydroxylgroup cause an even shallower penetration of the BS into themain body of the CD. This interpretation is supported by the lackof ROESY interactions between H3 and P18 in these complexesin contrast to the strong interactions between H3 and P18observed for the other complexes. As discussed below, confor-mational changes and the increased flexibility of m300 may alsocontribute to its extremely poor complexation properties.Enthalpy and Entropy. All investigated interactions were

exothermic, and a positive entropic contribution was seen for themajority of the complexes (see Table 2). A prominent feature ofthe complexes of HPβCDs with BSs was the strong enthalpy�entropy compensation associated with changes in the DS. Asthe DS increased, a decrease in ΔH� was observed, but this waslargely compensated by a corresponding increase in ΔS� suchthat ΔG� remained almost constant.12 This was interpreted asthe result of increased dehydration of the hydroxypropyl chainsand the hydrophobic parts of the BS that remained outside theCD cavity. As shown in the entropy�enthalpy compensationplot in Figure 7, which includes the HPβCD complexes, all of themβCD complexes seem to follow the same straight lines as theHPβCD complexes. This similar behavior suggests that the effectof the methyl substituents is the same as the effect of the HPsubstituents: dehydration of the hydrophobic parts of the BSanions that protrude from the cavity. However, due to the smallerarea of the methyl groups compared to the HP chains, eachmethyl substituent dehydrates a smaller area than the HPsubstituents and the increase in ΔH� and ΔS� with increasedDS is not as large as in the case of the HP substituents. Thisinterpretation may explain the complexation thermodynamics ofthe mβCDs except for m209, m212, and m300. Considering thehigh DS of these samples, they are expected to be found in theupper right corner of the ΔH�ΔS compensation plot, but them209 and m212 complexes lie in the middle and the complexesof m300 with GCDC and TCDC lie in the extreme lowerleft corner. This unexpected behavior of m209, m212, and in

Figure 7. Entropy�enthalpy compensation plot for all investigatedcomplexes including the complexes of HPβCDs with the same 6 BSsreported by Sch€onbeck et al.12 Complexes with natural βCD andmβCDs: GC and TC (0); TDC and GDC (O); TCDC and GCDC(Δ). Complexes with HPβCDs: GC and TC (9); TDC and GDC (b);TCDC and GCDC (2).

Figure 6. Measured stability constants for complexation with TC (9),GC (0), TDC (b), GDC (O), TCDC (2), and GCDC (Δ) as afunction of the average degree of substitution of the mβCDs.

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particular m300 suggests that other factors than the increaseddehydration discussed above contribute significantly to the largevariations in the thermodynamics of complexation.The very favorable ΔH� and very unfavorable ΔS� for com-

plexation of m300 with several guest molecules has beenpreviously observed.40,45 The large negative ΔH� is thought tostem from increased van der Waals contacts due to the increasedflexibility and the resulting better fit of m300 to the guestmolecule compared to natural βCD. For one type of guestmolecule, this leads to much larger stability constants,40,41 whilefor other guests the decrease in ΔH� is countered by an evenlarger decrease in ΔS� such that m300 forms weaker complexesthan the natural βCD.45 The BSs GCDC and TCDC clearlybelong to the latter group of guests. It is likely that thecomplexation-induced structural distortions lead to a good fitand large negative enthalpies. The accompanying large decreasein ΔS� might be caused by the restrictions in the movement ofthe methylglucose residues upon complexation. In its free formthe methylglucose residues of m300 are relatively free to tilt backand forth around the glycosidic bonds due to the lack of theH-bond network, but this movement is severely restricted in thecomplex. This may cause the large decrease inΔS� relative to theβCD inwhich theH-bond network on the rim of the CD is intact.However, according to the CIS of C1 and C4, m167 is also quiteflexible and undergoes large conformational changes upon com-plexation, but the complexes formed with this CD are character-ized by the least negative ΔH� and the largest positive ΔS� of allof the investigated mβCDs, so its behavior is exactly opposite tothat of m300. Apparently, the effects of the increased flexibility ofm167 are not strong enough to seriously affect the complexationthermodynamics, and the methyl substituents mainly contributeto ΔH� and ΔS� by increasing the dehydrated surface area.The purpose of the present study was to examine the relation-

ship between the structures of differently methylated βCDs andtheir abilities to form complexes with 6 different bile salts. Athorough NMR study determined the degree of methylation atthe 3 different sites of the CDs and established the structures ofthe complexes. By comparing this structural information with thethermodynamics of complexation, a well-founded structure�properties analysis was made. It is clear that it is crucial toknow both the positions of the methyl substituents and thenumber of substituents in order to rationalize the thermody-namic data, since the effects of the methyl substituents depend sostrongly on the site of substitution.

’CONCLUSION

Changes in the degree and pattern of methylation of the CDsaffect their interaction with all types of BS similarly. Methylsubstituents affect the complexation thermodynamics in thefollowing ways. (i) Substitution at O3 hinders complexation.This may be because the O3 methyl group points toward thecenter of the CD opening and thereby disturbs the intrusion ofthe guest molecule. (ii)Methyl groups at O2 point away from theopening but can still participate in hydrophobic interactions withthe guest and thereby favor complexation. (iii) Methyl substit-uents increase the hydrophobic surface area that is dehydratedupon complexation. This causes an increase inΔH� andΔS�, butthe effect on the complex stability is relatively small due toenthalpy�entropy compensation. This effect is very similar tothe effect of 2-hydroxypropyl substitutents.12 (iv) Eliminationof the H-bond network between secondary hydroxyl groups

increases the flexibility of the free CD. This allows a better fit ofthe CD to the guest, resulting in a more favorable ΔH�, but thefixation of the CD in the complex causes such a large loss ofentropy that the complexes of m300 are severely destabilized.

’ASSOCIATED CONTENT

bS Supporting Information. Selected mass spectra of CDsand selected ROESY NMR spectra of complexes; assignmenttables of some BSs and CDs and their complexation-inducedshifts; thermodynamic data for complexation of tauro-conju-gated bile salts with all CDs; representative ITC enthalpograms.This material is available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Phone:(þ45) 3643 3596. Fax: (þ 45) 3643 8242. E-mail:rhol@lundbeck.com.

’REFERENCES

(1) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875–1917.(2) Liu, L.; Guo, Q.-X. J. Inclusion Phenom. Macrocycl. Chem. 2002,

42, 1–14.(3) In Cyclodextrins and Their Complexes: Chemistry, Analytical

Methods, Applications; Dodziuk, H., Ed.; Wiley-VCH: Weinheim, 2006.(4) Uekama, K. Chem. Pharm. Bull. 2004, 52, 900–915.(5) Brewster, M. E.; Loftsson, T. Adv. Drug Delivery Rev. 2007,

59, 645–666.(6) Davis, M. E.; Brewster, M. E. Nat. Rev. Drug Discovery 2004,

3, 1023–1035.(7) Rao., C. T.; Pitha, J.; Lindberg, B.; Lindberg, J. Carbohydr. Res.

1992, 223, 99–107.(8) M€ullers, B. W.; Brauns, U. J. Pharm. Sci. 1986, 75, 571–572.(9) Yuan, C.; Jin, Z. Y.; Li, X. H. Food Chem. 2008, 106 (1), 50–55.(10) Thompson, D. O. Crit. Rev. Ther. Drug 1997, 14, 1–104.(11) Pattarino, F.; Giovannelli, L.; Giovenzana, G. B.; Rinaldi, M.;

Trotta, M. J. Drug Delivery Sci. Technol. 2005, 15 (6), 465–468.(12) Sch€onbeck, C.; Westh, P.; Madsen, J. C.; Larsen, K. L.; St€ade,

L. W.; Holm, R. Langmuir 2010, 26 (23), 17949–17957.(13) Holm, R.; Madsen, J. C.; Shi, W.; Larsen, K. L.; St€ade, L. W.;

Westh, P. J. Inclusion Phenom. Macrocycl. Chem. 2011, 69, 201–211.(14) Yong, C. W.; Washington, C.; Smith, W. Pharm. Res. 2008, 25

(5), 1092–1099.(15) Saenger, W. R.; Jacob, J.; Gessler, K.; Steiner, T.; Hoffmann, D.;

Sanbe, H.; Koizumi, K.; Smith, S. M.; Takaha, T. Chem. Rev. 1998, 98(5), 1787–1802.

(16) Alvaro, D.; Cantafore, A.; Attili, A. F.; Gianni Corrandini, S.; DeLuca, C.; Minervini, G.; Di Base, A.; Angelico, M. Comp. Biochem.Physiol. 1986, 83B, 551–554.

(17) Tan, X.; Lindenbaum, S. Int. J. Pharm. 1991, 74, 127–135.(18) Holm, R.; Nicolajsen, H. V.; Hartvig, R. A.; Westh, P.;

Østergaard, J. Electrophoresis 2007, 28, 3745–3752.(19) Ollila, F.; Pentik€ainen, O. T.; Forss, S.; Johnson, M. S.; Slotte,

J. P. Langmuir 2001, 17, 7107–7111.(20) 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.(21) Holm, R.; Shi, W.; Hartvig, R. A.; Askjær, S.; Madsen, J. C.;

Westh, P. Phys. Chem. Chem. Phys. 2009, 11, 5070–5078.(22) Liu, Y.; Zhang, Q.; Guo, D. S.; Zhuang, R. T.; Wang, L. H.

Thermochim. Acta 2008, 470 (1�2), 108–112.(23) 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.(24) Holm, R.; Hartvig, R. A.; Nicolajsen, H. V.; Westh, P.;

Østergaard, J. J. Inclusion Phenom. Macrocycl. Chem. 2008, 61, 161–169.

5841 dx.doi.org/10.1021/la200381f |Langmuir 2011, 27, 5832–5841

Langmuir ARTICLE

(25) Ono, N.; Hirayama, F.; Arima, H.; Uekama, K.; Rytting, J. H.Model analysis for oral absorption of a drug/cyclodextrin complexinvolving competitive inclusion complexes. J. Inclusion Phenom. Macro-cycl. Chem. 2003, 44, 93–96.(26) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114

(26), 10663–10665.(27) Cicero, D. O.; Barbato, G.; Bazzo, R. J. Magn. Reson. 2001, 148

(1), 209–213.(28) Hwang, T.-L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114,

3157–3159.(29) Nyberg, N. T.; Duus, J. O.; Sorensen, O. W. J. Am. Chem. Soc.

2005, 127 (17), 6154–6155.(30) Tongiani, S.; Velde, D. V.; Ozeki, T.; Stella, V. J. J. Pharm. Sci.

2005, 94 (11), 2380–2392.(31) Mucci, A.; Schenetti, L.; Vandelli, M. A.; Ruozi, B.; Salvioli, G.;

Forni, F. Supramol. Chem. 2001, 12, 427–433.(32) Vandelli, M. A.; Salvioli, G.; Mucci, A.; Panini, R.; Malmusi, L.;

Forni, F. Int. J. Pharm. 1995, 118, 77–83.(33) Mucci, A.; Vandelli, M. A.; Salvioli, G.; Malmusi, L.; Forni, F.;

Schenetti, L. Supramol. Chem. 1996, 7, 125–127.(34) Mucci, A.; Schenetti, L.; Salvioli, G.; Ventura, P.; Vandelli,

M. A.; Forni, F. J. Inclusion Phenom. Macrocycl. Chem. 1996, 26, 233–241.(35) Campredon,M.; Quiroa, V.; Thevand, A.; Allouche, A.; Pouzard,

G. Magn. Reson. Chem. 1986, 24, 624–629.(36) Barnes, S.; Geckle, J. M. J. Lipid Res. 1982, 23, 161–170.(37) Cooper, A.; Nutley, M. A.; Camilleri, P. Anal. Chem. 1998,

70, 5024–5028.(38) Harata, K.; Uekama, K.; Otagiri, M.; Hirayama, F. J. Inclusion

Phenom. Macrocycl. Chem. 1984, 1 (3), 279–293.(39) Schneider, H.-J.; Hacket, F.; R€udiger, V. Chem. Rev. 1998, 98,

1755–1785.(40) Kano, K.; Nishiyabu, R.; Doi, R. J. Org. Chem. 2005, 70 (9),

3667–3673.(41) Kano, K.; Nishiyabu, R.; Asada, T.; Kuroda, Y. J. Am. Chem. Soc.

2002, 124 (33), 9937–9944.(42) Liu, Y.; Shi, J.; Guo,D. S. J. Org. Chem.2007, 72 (22), 8227–8234.(43) Tan, Z. J.; Zhu, X. X.; Brown, G. R. Langmuir 1994, 10,

1034–1039.(44) Momose, T.; Yamaguchi, Y.; Iida, T.; Goto, J.; Nambara, T.

Lipids 1998, 33 (1), 101–108.(45) Kano, K.; Tamiya, Y.; Hashimoto, S. J. Inclusion Phenom. Mol.

1992, 13 (3), 287–293.(46) Szejtli, J. Chem. Rev. 1998, 98, 1743–1753.