Methylated β-Cyclodextrins: Influence of Degree and Pattern of

ARTICLE pubs.acs.org/Langmuir

Methylated β-Cyclodextrins: Influence of Degree and Pattern of Substitution on the Thermodynamics of Complexation with Tauro- and Glyco-Conjugated Bile Salts Christian Sch€onbeck,†,‡ Peter Westh,† Jens Christian Madsen,§ Kim Lambertsen Larsen,|| Lars Wagner St€ade,|| and Rene 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 of substitution affects the binding. The structures of the CDs were determined by mass spectrometry and NMR techniques, and the structures of the inclusion complexes were characterized from the complexation-induced shifts of 13C nuclei as well as by 2D ROESY NMR. Thermodynamic data were generated using isothermal titration calorimetry. The structure
properties analysis showed that methylation at O3 hinders complexation by partially blocking the cavity entrance, while methyl groups at O2 promote complexation by extending the hydrophobic cavity. Like in the case of 2-hydroxypropylated cyclodextrins, the methyl substituents cause an increased release of ordered water from the hydration shell of the bile salts, resulting in a strong increase in both the enthalpy and the entropy of complexation with increased number of methyl substituents. Due to enthalpy
entropy compensation the effect on the stability constant is relatively limited. However, when all hydroxyl groups are methylated, the rigid structure of the free cyclodextrin is lost and the complexes are severely destabilized due to very unfavorable entropies.

’ INTRODUCTION Cyclodextrins (CDs) are ring-shaped molecules often described as truncated cones. Due to their hydrophilic outer surface and hydrophobic inner cavity they are able to form inclusion complexes with a large variety of predominantly hydrophobic guest molecules.1,2 This makes CDs useful for many applications,3 especially within pharmaceutical4 and food sciences. At present there exist 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 therefore diameter of the cavity, which increases in size from R to γ. They present 18, 21, and 24 modifiable hydroxyl groups, respectively, distributed with 2/3 on one rim (often termed the wide rim) and 1/3 on the other (termed the narrow rim). Great efforts have r 2011 American Chemical Society

been put into developing modified CDs with altered properties compared to the native CDs and in controlling the degree and pattern of the substitution. Hence, a large number of modified CDs exist in which various substituents are attached to the oxygens in the 2-, 3- and 6-positions (see Figure 1). Introduction of substituents alters both the physicochemical properties of the CDs and the ability to form complexes.7
13 Thus, if the effects of the different substituents are well understood, one might tailor CDs for specific guest molecules and for specific purposes in order to achieve optimal properties. Received: January 28, 2011 Revised: April 5, 2011 Published: April 21, 2011 5832

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Figure 1. Numbering of the nuclei in methylated βCD.

The primary driving forces for complexation with CDs are believed to arise from hydrophobic and van der Waals interactions, with contributions from other forces such as hydrogen bonds, if present.2 Recently, a systematic study of 2-hydroxypropylated βCDs (HPβCDs) was published in which the effects of the degree of substitution (DS) on the thermodynamics of complexation with bile salts (BSs) was investigated.12 Although the DS had only a minor influence on the association constants, it had a strong influence on the enthalpy and entropy of complexation, both increasing linearly with increasing DS. This was interpreted as the result of an increased release of ordered water from the hydration shells of the guest molecules and the hydroxypropyl substituents. In addition to HPβCD, methylated CDs are among the most commonly used CDs. Therefore, a systematic investigation of how the degree and pattern of substitution affect the ability of methylated βCD (mβCD) to form inclusion complexes will provide useful information for the utilization of this class of CDs. Furthermore, an investigation of the effects of the degree and pattern of methylation on the thermodynamic parameters, ΔH° and ΔS°, associated with complexation will reveal some of the effects driving the interaction. There are several structural differences between the methyl and HP substituents that might affect the complexation properties of the CD: (1) The HP chain contains a hydroxyl group that is expected to interact with the surrounding water molecules and may also participate in intramolecular hydrogen bonds, as suggested by molecular dynamics simulations,14 (2) the HP chain is more flexible, and (3) the HP chain has a larger surface area and may therefore interact with and dehydrate larger parts of the hydrophobic guest molecule. Another interesting aspect is the commercial availability of mβCDs spanning a larger range of DS. The largest DS of the investigated HPβCDs was 1.06 (on average 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 the HPβCDs was O2, but the larger DS of the mβCD forces a significant fraction of the substituents to be located at the two other sites on the glycopyranose units, i.e., O3 and O6. It was not possible to precisely determine the substitution pattern of the HPβCDs, but in the present work the substitution patterns of the mβCDs are determined, and this enables a discussion of the effects of having substituents at the various sites of the CD. This may provide useful information about the thermodynamic importance of the site of substitution. A large number of studies investigating the complexes of mβCDs with various guest molecules are published in the literature. The majority of these studies investigate dimethylated or trimethylated CDs where two or all of the substitution sites (O2, O3, and O6) are completely methylated. Only a small number of studies focus on randomly methylated βCDs (RAMEB) where each of the

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3 sites is only partially methylated. This lack of attention might be due to the fact that all RAMEB samples are mixtures of many isomers and that the samples need to be characterized with respect to the degree and pattern of substitution before a detailed analysis and discussion concerning the effects of the substituents can be made. However, RAMEBs are widely used as pharmaceutical solubilizers,5 and it is therefore important to understand the structural factors governing the behavior of this class of mβCDs. Also, from an academic point of view RAMEBs are interesting since partial methylation allows the intramolecular hydrogen-bond network15 to be partially retained. Thus, it might be possible to observe the steric and hydrophobic effects of the methyl groups while avoiding the effects of the complete elimination of the hydrogen-bond network that occurs when O2 and O3 are completely methylated. In the present work both dimethylated and trimethylated βCDs as well as a range of RAMEBs are studied, and all CD samples are thoroughly characterized with respect to the average number and position of substituents and the distribution in the number of substituents within each sample. To our knowledge, such a comprehensive study of the complex forming capabilities of well-characterized RAMEBs has not previously been conducted. In order to clarify the effects of methyl substituents, the present study investigates the complexing abilities of methylated βCDs having a wide range of DS by use of isothermal titration calorimetry (ITC). This technique provides precise values of association constants, K, as well as direct determination of enthalpies (ΔH°) and entropies (ΔS°) of complexation and thereby provides useful thermodynamic insight into CD chemistry. The guest molecules in the present study are 6 different bile salts (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 the release profile following oral administration of a compound in a CD complex.4,25 In order to assist a structural interpretation of the thermodynamic parameters, the complexes are structurally characterized by ROESY-NMR and the complexation-induced shifts (CIS) of 1H and 13C NMR peaks.

’ EXPERIMENTAL SECTION Materials. Sodium salts of the bile acids taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), glycocholate (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 nominal average 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 a generous gift from Roquette (Le Strem, France). A single sample of randomly methylated βCD with no specification of DS was purchased from Sigma-Aldrich along with two samples of heptakis(2,6-di-Omethyl)-βCD and a single sample of heptakis(2,3,6-tri-O-methyl)βCD. In the following, the CD samples will be named according to the DS determined experimentally by mass spectrometry. The names, as well as the degree and pattern of substitution, appear from Table 1. MilliQ water was used for all solutions, and all chemicals were of analytical grade 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-4hydroxycinnamic acid (CCA) on a Reflex III (Bruker Daltonics, Bremen, Germany). 10 mM solutions of the mβCD samples dissolved in a 1:4 (v/v) mixture of nitro-cellulose and saturated CCA in an aqueous solution of 0.1% trifluoroacetic acid and 80% acetonitrile were applied directly to the MALDI target plate. 5833

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

Table 1. Average Degree of Substitution and Distribution of Substituents measured DS (MS)

measured DS (1H NMR)

DS(O2)a

DS(O2)b

DS(O3)b

DS(O6)b

0.6

0.67

0.44

0.36

0.38

0.19

0.10

0.57

0.69

0.63

0.52

0.52

0.12

0.05

1.2

1.17

1.03

0.55

0.59

0.33

0.25

1.63

1.28

0.65

0.62

0.34

0.67

CD

reported DS from supplier

m067 m069 m117 m163 m167 m209

1.8 2

1.67 2.09

1.50 1.90

0.64 1.00

0.63 1.00c

0.37 0.09c

0.67 1.00c

m212

2.16

2.12

1.95

1.00

1.00c

0.12c

1.00c

1.00

d

d

1.00d

m300 a

3

3.00

2.99

1.00

1.00

0 b

DS(O2) is determined from peak areas of H1 and H1 . Determined from peak areas of the methyl carbons in combination with DS from MS. c There are 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 at O3. d All spectra show that the sample consists of heptakis(2,3,6-tri-O-methyl)-βCD.

NMR Spectroscopy. For characterization of the CD samples, standard 1H spectra, attached proton test (APT), and two-dimensional HSQC,26 COSY, and HMBC27 spectra were recorded at 25 °C on a Bruker Avance-600 NMR spectrometer operating at 14.1 T and equipped with a cryogenically cooled probe. The experiments were performed on 10 mM CD samples in D2O. For the structural characterization of the complexes, 2D ROESY28 spectra were recorded in addition to the above-mentioned experiments. The concentrations of CD and BS in the D2O solutions were both 10 mM. For the assignment of the free BS anions GC, GDC and GCDC, H2BC29 spectra were recorded in addition to APT, HSQC, and HMBC spectra. To avoid micellization of the 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 in 50 mM phosphate buffer pH 7.1. The reaction cell of the calorimeter was filled with BS solutions ranging from 0.25 to 1 mM and was titrated with CD solutions in the concentration range 2.5
20 mM. The higher the stability constants, the more diluted the solutions. Concentrations of the CD solutions were calculated from the average molecular weight of each CD sample as determined by mass spectrometry. The titrations were performed in triplicate on a Microcal 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 CD solutions into buffer solution. The measured heats of dilution were subtracted from the titration heats to yield the heats of complexation. The titration curves were fitted using MicroCal’s ITC data analysis application to the Origin software package (version 7.0) assuming a single set of identical and independent binding sites. This yields the complexation stoichiometry (n), the molar enthalpy of complexation (ΔH°), and the stability constant (K). The stability constant and molar enthalpy enables calculation of the standard Gibbs free energy of complexation (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-TOF mass spectrometry revealed that only the m300 sample was monodisperse. All other samples consisted of a mixture of CDs having different DS. The randomly methylated CD samples were 5834

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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 and vertical projection, respectively. The subscript Nsub denotes the directly substituted carbon (or the proton attached to this carbon), while the neighboring 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.

the most polydisperse with 6
9 CDs of different masses with a normal distribution around the average molecular mass. The largest peaks in the mass spectra of m209 and m212 corresponded to heptakis(2,6-di-O-methyl)-βCD, but significant amounts of higher substituted CDs were also present. All mass spectra are found in the Supporting Information. From the peak intensities in the mass spectra the average DS were calculated as DS ¼

∑i Ii 3 DSi ∑i Ii

where Ii denotes the intensity of the ith peak and DSi denotes the degree of substitution corresponding to the ith peak. The calculated 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 the found one. The distribution of masses and the average DS may be determined by MS, but the pattern of substitution must be determined by NMR. From analysis of 1D 1H NMR, 1D 13C NMR, and 2D HSQC and HMBC spectra, the peaks in the 1D spectra were assigned as shown in Figures 3 and 4 and the Supporting Information. In agreement with previous observations,13,30 the directly substituted carbons (C2, C3, and C6) were shifted ∼10 ppm downfield relative to their unsubstituted counterparts while the neighboring carbons experience upfield shifts between 1 and 3 ppm. Protons on directly substituted carbons are shifted upfield by 0.16
0.27 ppm, while protons on neighboring carbons are shifted downfield by 0.06
0.19 ppm. These shifts are observed in all of the methylated CDs with the exception of m300, where the chemical shifts of many peaks differ somewhat from 5835

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Figure 5. (A) Suggested structure of the 1:1 complexes between GCDC and all CDs. (B) Suggested structure of the 2:1 CD:BS complexes between GDC and all CDs except m300. The cyclodextrins are represented by their cavities whose dimensions more or less correspond to the ones given by Szejtli.46

the corresponding peaks in the other CDs. This suggests a distortion of the CD ring induced by disruption of intramolecular hydrogen bonds. Judging from the chemical shifts, this distortion does not happen gradually as the DS increases but only when all hydroxyl groups are substituted. The average degree of substitution may be quantified from the proton peak areas. As seen on Figure 4, the only peaks that are not overlapping are H1 and H10 , so the DS can be determined by comparing the sum of these two peaks (which integrates 1 proton) to the summed peak areas of all other protons. The results are listed in Table 1. The DS obtained from 1H NMR are consistently lower than the ones obtained from MS. From the mass spectra it is clear that the DS of m209 and m212 must be larger 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 also the degree of substitution on O2 [DS(O2)] may be quantified from the relative areas of H1 and H10 and are also shown in Table 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 may be compared to each other and the area of a substituted carbon may be compared to the corresponding unsubstituted carbon. Thus, the degree of substitution on each site may be calculated from the areas of substituted and unsubstituted carbon peaks. However, due to partially overlapping peaks, this is not possible in all cases. A better method may be to calculate the relative distribution of substituents by comparing the areas of the methyl carbons. Once the fraction of the methyl groups attached to C2 is known, DS(O2) may be calculated by multiplying with the total DS. DS(O3) and DS(O6) are calculated in the same way and are listed in Table 1. From these results it is apparent that a wide range of the number and positions of substituents is evaluated in the present study.

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Structural Characterization of Complexes. A more qualified interpretation of the measured thermodynamic parameters can be obtained if the structures of the complexes are established. For structural characterization of the complexes, two different NMR methods were employed. The first is 2D ROESY in which protons that are spatially close give rise to off-diagonal crosspeaks whose intensities depend on the distance between the protons. The cross-peaks between CD protons and BS protons will 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 molecule are replaced by the hydrophobic interior of the CD, the peaks of the BS nuclei experiencing this change in environment will move in the NMR spectrum. This complexation-induced shift (CIS) can be determined for 1H and 13C nuclei and has previously been used to determine the inclusion site of the guest molecule.13,31
34 These two methods were used to investigate the structures of the 12 complexes formed between the 3 BSs GC, GDC, and GCDC 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 in agreement with the literature13,35,36 and are shown together with a table of all 13C CIS and selected ROESY spectra in the Supporting Information. Complexes with GCDC. From both the ROESY spectra and the CIS of the BS 13C it is clear that all 4 investigated CDs form similar 1:1 inclusion complexes with GCDC. The CDs encapsulate parts of the C and D rings of the steroid structure and part of the conjugated side chain of GCDC, as previously reported.13,21 It is apparent that the GCDC nuclei 12, 13, 16, 17, 18, and 20
24 all have large CIS, strongly suggesting that these nuclei are included within the CDs. Interestingly, nuclei 11 and 15 exhibit large CIS in the complex with m300 but not in the complexes with βCD and m209. This suggests that the methyl substituent at O3, which is present in m300 but not in βCD and m209, extends the hydrophobic cavity to also include nuclei 11 and 15. The polydisperse m167, which is partially substituted at O3, also induces 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 and H5 and the BS protons (denoted with a P to distinguish them from the CD protons). H3 is located close to the wide opening of the CD, while H5 is located closer to the narrow opening. The strong ROESY interactions between H3 and P18 and between H5 and P21 suggest that GCDC enters the CDs from the wide opening, as previously observed for the natural βCD.21 In addition, H5 interacts with protons on the side chain and the D ring, while H3 interacts with protons on the C and D rings. From these observations and the 13C CIS described above, the complex structure shown in Figure 5A is deduced for the complexes between 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 peaks completely disappear in the noise. It is apparent that it is the nuclei 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. Since the broadened nuclei are also the ones that experience the largest 13 C CIS, it may be concluded that the broadening is due to inclusion of GC in the CD and that the structure of the complex is quite similar to the ones formed with GCDC. The structure is supported by the ROESY spectra and is consistent with 5836

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Langmuir 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, but the 13C CIS are all small, and no correlation peaks between H3 and H5 and the GC protons are observed in the ROESY spectrum. This indicates a very limited or no inclusion of GC into m300. Complexes with GDC. Peak broadening was also observed in the APT spectra of the GDC complexes. In contrast to the GC complexes, not only are the nuclei at the C and D rings and the side chain broadened but also most of the nuclei at the A and B rings are broadened. The nuclei least affected by broadening are those in the middle of the BS, e.g., numbers 8, 9, and 10. Almost all nuclei have relatively large CIS, and together with the peak broadening pattern, this suggests that the CDs bind to both ends of 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 CDs to the A and B rings of the BS is confirmed by the ROESY spectra, in which there are many correlation peaks between the interior CD protons H3 and H5 and several protons on the A and B rings of GDC. H3 tends to interact strongest with P4 and P6, while H5 interacts strongest with P2 and P4, thereby confirming the structure in Figure 5B, in which the BS enters both CDs through the wide openings. Again, the complex GDC/m300 might be an exception since the ROESY cross-peaks are few, weak, and hard to assign. It is clear, however, that an inclusion complex is formed since ROESY interactions are observed between the interior CD protons H3 and H5 and a number of BS protons, including a strong interaction between H5 and P21 and a weak interaction between H3 and P19. Interestingly, in contrast to all of the other inclusion complexes, no interaction between H3 and P18 is observed. The binding to a second binding site or formation of 2:1 CD: BS complexes has previously been observed for complexes between natural βCD and this type of BS that has no hydroxyl group on C7 and therefore permits inclusion of the A and B rings.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 that the methyl groups on O2 (2-CH3) of the permethylated CDs point away from the center of the CD cavity, while the 3-CH3 groups point toward the center of the cavity opening.38 The present study strongly suggests that this orientation of the methyl groups 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 P19 or no BS protons at all. Furthermore, large 13C CIS are seen for 3-CH3 in the complexes of m300 with GDC and GCDC, but the other 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) when complexed with one of the BS. The CIS of these two nuclei vary greatly between the CDs and increase in the order βCD < m209 < m167 , m300, with the CIS in m300 reaching 3.26 and 4.81 ppm for C1 and C4, respectively. The C1 and C4 carbons participate in the glycosidic bonds between the methylglucose residues, and the large CIS of these nuclei probably reflects large conformational changes upon complexation, in which the tilt angles of the methylglucose residues are significantly changed. The flexibility of the CDs increases with increased substitution due to disruption of the H-bond network between the secondary

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hydroxyl groups, and this explains why the CIS of C1 and C4 increases in the observed order.39 Apparently, m300 undergoes large conformational changes upon complexation, whereas the natural βCD has a much more rigid structure. Interestingly, these large CIS are not observed for complexation of m300 with GC, adding to the assumption that these two molecules do not form an inclusion complex. The observed CIS for complexation of GCDC with βCD and m300 are quite similar to those observed for complexation of neutral and charged porphyrins with the same two CDs.40,41 Despite the different characters of the guest molecules they induce similar CIS on the 13C nuclei of the CDs, and this strongly suggests that it is the conformational 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 CDs and 6 BS. Each CD sample, with the exception of the natural βCD and m300, is actually a mixture of many different mβCDs of various masses and substitution patterns. This means that each enthalpogram represents not just a single complexation equilibrium but many competing equilibria, each characterized by an individual equilibrium constant. An exact model of such system consisting of many linked equilibria would be too complex to provide meaningful fits to the enthalpograms. However, as evidenced by the mass spectra there is a relatively narrow distribution in the number of substituents per CD within each sample. Therefore, it might be assumed that all CDs within a sample have somewhat similar affinities toward the BS. In such case the multiple binding equilibria will essentially behave like a single equilibrium describing the complexation between the BS and a single “average” mβCD characterized by the average DS of the sample. On the basis of this assumption the enthalpograms were fitted with a model based on one set of identical and independent binding sites. This model provided good fits to all enthalpograms and yielded stoichiometries of about 1, thereby validating the above assumption. Representative enthalpograms are shown in the Supporting Information. Although the NMR study of the complex structures provides evidence of a secondary binding site on the GDC anions, this secondary interaction is apparently too weak to be detected by the method used in the ITC studies. If ΔG° and ΔH° for the secondary binding site are small compared to the primary site, the heats produced by binding to the secondary site are so small that they “vanish” in the much larger heats produced by binding to the primary site. The titrations of GC/TC and GDC/TDC with m300 all produced very small heats. Compared to these small heats the enthalpy of dilution of m300 is relatively large and the subtraction of the enthalpy of dilution therefore introduces relatively large errors on the corrected reaction enthalpies. Although the one set of sites model fits the reaction enthalpies pretty well the enthalpograms show no real signs of saturation and it is concluded that the enthalpies of complexation and/or the stability constants are too small to be correctly determined by ITC. Titrations of GC and TC with m300 conducted by Liu et al.42 yielded very small stability constants on the order of 200 M
1. In accordance with the discussions in the NMR section, it may be concluded that GDC/TDC and GC/TC form weak inclusion complexes with m300. Thermodynamics of Complexation. In addition to the stoichiometries, the fitting of the model to the enthalpograms yielded values for the stability constant (K) and the molar enthalpy of complexation (ΔH°), from which the standard Gibbs free energy 5837

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

GDC

GCDC

host

K (M
1)

ΔG° (kJ/mol)

ΔH° (kJ/mol)

ΔS° (J/mol 3 K)

βCD

2876 ( 81


19.7 ( 0.1


25.7 ( 0.2


19.9 ( 0.9

m069 m067

3464 ( 77 2902 ( 51


20.2 ( 0.1
19.8 ( 0.0


20.0 ( 0.2
19.2 ( 0.4

0.9 ( 1.0 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 20.3 ( 0.2

m167

1742 ( 18


18.5 ( 0.0


12.5 ( 0.1

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

βCD m069

3075 ( 71 7374 ( 111


19.9 ( 0.1
22.1 ( 0.0


31.3 ( 0.2
22.3 ( 0.0


38.2 ( 0.8
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 βCD

NA b 139 733 ( 4852

NA b
29.4 ( 0.1

NA b
31.0 ( 0.2

NA b
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 m300

151 833 ( 3516 2671 ( 350


29.6 ( 0.1
19.5 ( 0.3


27.0 ( 1.0
34.8 ( 2.4

8.8 ( 3.2
49.2 ( 7.8

a

A similar table containing the thermodynamic parameters for the tauro-conjugated bile salts is found in the Supporting Information. The listed uncertainties are calculated as the standard deviation of 3 different experiments. b The association between the CD and the BS is too weak to be reliably determined by ITC.

(ΔG°) and the molar entropy of complexation (ΔS°) can be calculated. These are shown in Table 2, and representative fits are shown in the Supporting Information. The relationship between the BS structures and the stabilities of complexes formed with βCD and modified βCDs has previously been described.13,17,18,21,43,44 The lack of a hydroxyl group on C12 allows a deeper inclusion of GCDC and TCDC, and consequently, the stability constants are much larger than for the other BSs, which possess a hydroxyl group at C12. The presence of a hydroxyl group at C7 has an analogous but less pronounced influence on the stability constants as seen by the larger stability constants of complexes with GDC/TDC compared to complexes with GC/TC. The type of amino acid conjugation only has a minor influence in that the glyco-conjugated BSs have slightly larger stability constants than the tauro-conjugated ones. These general trends are also seen for the methylated βCDs in the present study with the small exception that m209 and m212 form 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. Unlike the HPβCDs,12 in which the stability constant systematically decreased with increasing DS, no simple relation between the stability constant and the DS was seen for the investigated mβCDs. However, binding to all types of BSs is similarly affected by the degree of substitution. Small DS (∼0.7) increases the

binding to all BSs relative to the natural βCD, but further methylation decreases the affinity with a local minimum at DS ≈ 1.7. Then the stability constants increase for m209 and m212 and decrease significantly in the case of m300, which has very poor binding properties compared to the other CDs. To explain these observations it is necessary to take into account the position of the methyl substituents instead of just considering the total number of substituents. A methyl substituent affects the complexation in different ways depending on whether it is situated at O2, O3, or O6. The substitution patterns of the mβCDs used in the present study are presented in Table 1. The obtained stability constants suggest that methylation at O3 severely hinders complexation while methylation at O2 improves complexation. At low DS (∼0.7), most of the methyl groups are situated at O2 and this improves the binding relative to the natural βCD. This increase is more pronounced for m069 than for m067, both having DS ≈ 0.7, since the former has more methyl groups at O2 and fewer at O3. At higher DS, the increase in the number of methyl groups mainly takes place at O3 and O6 and this causes the stability constants to decrease. However, m209 and m212 are both completely methylated at O2 and only slightly methylated at O3, and therefore, they bind more strongly to the BSs than m163 and m167, in which more of the substituents are at O3. m212 has a slightly higher DS than 5838

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Figure 7. Entropy
enthalpy compensation plot for all investigated complexes including the complexes of HPβCDs with the same 6 BSs reported by Sch€onbeck et al.12 Complexes with natural βCD and mβCDs: 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 a function of the average degree of substitution of the mβCDs.

m209, and since the O2 and O6 sites are probably completely substituted, the extra methyl groups on m212 must be situated at O3. This agrees well with its lower binding properties. The additional full methylation at O3 dramatically reduces the complexation properties of m300. A comparison of m117 with m167 and m169 indicates that methylation at O6 reduces the stability constant. However, the negative influence of methylation at O6 seems to be relatively weak and does not significantly disturb the good binding properties of m209 and m212 resulting from the large number of substituents at O2 and the small number 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 is tempting to suggest that these methyl groups hinder inclusion by partially blocking the cavity entrance.10 Methyl groups at O2, which point away from the entrance, do not hinder inclusion and may favor inclusion by extending the hydrophobic cavity of the CDs.10 The low affinity of m300 toward GC/TC and GDC/ TDC could very well be caused by unfavorable interactions between the hydroxyl group on C12 on the BS and the methyl groups on O3 of m300. As discussed above, the hydroxyl group on C12 of the BS hinders a deep inclusion of the BS and causes a

strong decrease in the stability constant. The presence of methyl groups on all O3s and their interactions with the C12 hydroxyl group cause an even shallower penetration of the BS into the main body of the CD. This interpretation is supported by the lack of ROESY interactions between H3 and P18 in these complexes in contrast to the strong interactions between H3 and P18 observed for the other complexes. As discussed below, conformational changes and the increased flexibility of m300 may also contribute to its extremely poor complexation properties. Enthalpy and Entropy. All investigated interactions were exothermic, and a positive entropic contribution was seen for the majority of the complexes (see Table 2). A prominent feature of the complexes of HPβCDs with BSs was the strong enthalpy
entropy compensation associated with changes in the DS. As the DS increased, a decrease in ΔH° was observed, but this was largely compensated by a corresponding increase in ΔS° such that ΔG° remained almost constant.12 This was interpreted as the result of increased dehydration of the hydroxypropyl chains and the hydrophobic parts of the BS that remained outside the CD cavity. As shown in the entropy
enthalpy compensation plot in Figure 7, which includes the HPβCD complexes, all of the mβCD complexes seem to follow the same straight lines as the HPβCD complexes. This similar behavior suggests that the effect of the methyl substituents is the same as the effect of the HP substituents: dehydration of the hydrophobic parts of the BS anions that protrude from the cavity. However, due to the smaller area of the methyl groups compared to the HP chains, each methyl substituent dehydrates a smaller area than the HP substituents and the increase in ΔH° and ΔS° with increased DS is not as large as in the case of the HP substituents. This interpretation may explain the complexation thermodynamics of the mβCDs except for m209, m212, and m300. Considering the high DS of these samples, they are expected to be found in the upper right corner of the ΔH
ΔS compensation plot, but the m209 and m212 complexes lie in the middle and the complexes of m300 with GCDC and TCDC lie in the extreme lower left corner. This unexpected behavior of m209, m212, and in 5839

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Langmuir particular m300 suggests that other factors than the increased dehydration discussed above contribute significantly to the large variations in the thermodynamics of complexation. The very favorable ΔH° and very unfavorable ΔS° for complexation of m300 with several guest molecules has been previously observed.40,45 The large negative ΔH° is thought to stem from increased van der Waals contacts due to the increased flexibility and the resulting better fit of m300 to the guest molecule compared to natural βCD. For one type of guest molecule, this leads to much larger stability constants,40,41 while for other guests the decrease in ΔH° is countered by an even larger decrease in ΔS° such that m300 forms weaker complexes than the natural βCD.45 The BSs GCDC and TCDC clearly belong to the latter group of guests. It is likely that the complexation-induced structural distortions lead to a good fit and large negative enthalpies. The accompanying large decrease in ΔS° might be caused by the restrictions in the movement of the methylglucose residues upon complexation. In its free form the methylglucose residues of m300 are relatively free to tilt back and forth around the glycosidic bonds due to the lack of the H-bond network, but this movement is severely restricted in the complex. This may cause the large decrease in ΔS° relative to the βCD in which the H-bond network on the rim of the CD is intact. However, according to the CIS of C1 and C4, m167 is also quite flexible and undergoes large conformational changes upon complexation, but the complexes formed with this CD are characterized by the least negative ΔH° and the largest positive ΔS° of all of the investigated mβCDs, so its behavior is exactly opposite to that of m300. Apparently, the effects of the increased flexibility of m167 are not strong enough to seriously affect the complexation thermodynamics, and the methyl substituents mainly contribute to ΔH° and ΔS° by increasing the dehydrated surface area. The purpose of the present study was to examine the relationship between the structures of differently methylated βCDs and their abilities to form complexes with 6 different bile salts. A thorough NMR study determined the degree of methylation at the 3 different sites of the CDs and established the structures of the complexes. By comparing this structural information with the thermodynamics of complexation, a well-founded structure
properties analysis was made. It is clear that it is crucial to know both the positions of the methyl substituents and the number of substituents in order to rationalize the thermodynamic data, since the effects of the methyl substituents depend so strongly on the site of substitution.

’ CONCLUSION Changes in the degree and pattern of methylation of the CDs affect their interaction with all types of BS similarly. Methyl substituents affect the complexation thermodynamics in the following ways. (i) Substitution at O3 hinders complexation. This may be because the O3 methyl group points toward the center of the CD opening and thereby disturbs the intrusion of the guest molecule. (ii) Methyl groups at O2 point away from the opening but can still participate in hydrophobic interactions with the guest and thereby favor complexation. (iii) Methyl substituents increase the hydrophobic surface area that is dehydrated upon complexation. This causes an increase in ΔH° and ΔS°, but the effect on the complex stability is relatively small due to enthalpy
entropy compensation. This effect is very similar to the effect of 2-hydroxypropyl substitutents.12 (iv) Elimination of the H-bond network between secondary hydroxyl groups

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

’ ASSOCIATED CONTENT

bS

Supporting Information. Selected mass spectra of CDs and selected ROESY NMR spectra of complexes; assignment tables of some BSs and CDs and their complexation-induced shifts; thermodynamic data for complexation of tauro-conjugated 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: [email protected].

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