Hydroxypropyl-Substituted β-Cyclodextrins: Influence of Degree of

The NMR experiments were carried out at 25 °C on a Bruker Avance-600 NMR ..... 3.6 kJ/mol to the enthalpy of complexation and also 3.6 kJ/mol to TΔS...
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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 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, 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 form inclusion complexes with six different bile salts, found within the intestinal tracts of rats, dogs, and humans, was studied by isothermal titration calorimetry. The composition and molecular structure of the cyclodextrin samples were characterized by MALDI-TOF mass spectrometry together with 1D and 2D-NMR, and some of the complexes were studied by 2D ROESY NMR. The stability and structure of the complexes were mainly determined by the position of hydroxyl 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 a pronounced enthalpy-entropy compensation was observed. These observations are interpreted as an increased release of ordered water from the hydration shells of the bile salts, caused by the hydroxypropyl substituents on the rim of the CD. It is estimated that each CD hydroxypropyl substituent dehydrates a hydrophobic surface area of approximately 10 A˚2.

Introduction Cyclodextrins (CDs) are cyclic oligosaccharides with the shape of a truncated cone. Owing to their hydrophilic outer surface and their lipophilic cavity, they are able to form inclusion complexes with hydrophobic guests in aqueous solution. Thus, they may act as solubilizers of water insoluble drugs and are used as drug carriers among other things.1-13 The most common CDs are R, β, and γ-cyclodextrins, consisting of six, seven, and eight glycopyranose units, respectively. The size of the cavity increases with the number of glucopyranose units, and this has a large influence on their complexation capabilities, since a strong interaction requires a good fit between the guest molecule and the CD cavity.14 Thus, there is a preference for the inclusion of guest molecules or parts of guest molecules that match the size of the cavity. *To whom correspondence should be addressed. Email: rhol@lundbeck. com. Tel.: (þ45) 3643 3596. Fax: (þ45) 3643 8242. Address: H.Lundbeck A/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.

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βCD has a relatively low aqueous solubility that severely limits its uses. Substituting the hydroxyl groups at the rim of the βCD results in a dramatic increase in aqueous solubility, which might be due to interruption of intramolecular hydrogen bonds between hydroxyl groups on the rims of the βCD.15 The high solubility of 2-hydroxypropylated βCD (HPβCD) makes it a good alternative to the natural βCD, and it is used in several marketed pharmaceutical products.3,4 However, modifications of the natural CDs not only alter the physicochemical properties of the CDs and their complexes, but also the complexation abilities of the CDs.16-22 A number of studies have investigated how the stability constants of the complexes are affected by the degree of substitution (DS) of HPβCD.17-21 This strongly depends on the type of guest. For some guests, the stability constant increases with increasing DS, while for others it decreases. Generally, “large spherical” guests, exemplified by phenolphthalein, form weaker complexes with increasing DS, while smaller guests form stronger complexes as the DS increases.19,20 The substituents are thought to affect complexation in two ways: (1) steric blockage of the cavity openings by the substituents, resulting in a lower stability constant and (2) extension of the CD cavity, leading to increased hydrophobic interactions with the hydrophobic parts of the guest (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.

Published on Web 11/03/2010

DOI: 10.1021/la103124n

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by a range of experimental techniques;30-43 however, no systematic investigations exist on the influence of CD substitution on the thermodynamics of the interaction. The purpose of the present study is, therefore, to examine the thermodynamics of the interaction between bile salts and HPβCDs in order to elucidate the effects of HP-substitutions and to obtain a more general understanding of the driving forces behind the inclusion complex formation between cyclodextrins and hydrophobic guest molecules. This may assist in a rational choice of modified βCD for oral drug delivery.

Experimental Section

Figure 1. One of the seven glycopyranose units of substituted

βCD.

molecule, resulting in a higher stability constant.12 Not only is the degree of substitution important but the pattern of substitution also influences complexation properties.19,20 As sketched in Figure 1, each of the glycopyranose units may be substituted at three different sites: O2, O3 and O6. O2 and O3 are situated on the rim of the wider opening of the CD, while O6 is on the rim of the narrow opening. It is speculated that the presence of substituents at O2 and O6 may extend the hydrophobic cavity, whereas substituents at O3 and O6 may narrow the cavity openings12 and molecular dynamics simulations of HPβCD in water show that HP groups at the O2 sites expand the wider cavity opening.23 While several authors have investigated the influence of the DS on the stability constant, K, only a few studies have systematically investigated how variations in the DS affect other thermodynamic parameters.18,24-26 In those studies, the stability constants of complexes formed between various guest molecules and sulfobutylether-βCDs and HPβCDs of different DS were obtained, as well as the thermodynamic parameters ΔH° and ΔS° for complex formation. Knowing the latter two parameters allows for a more detailed interpretation of the role that the substituents play in the stability of the complexes and a better understanding of the different contributions to the stability of the complexes. In the present study, six bile salts (BS) (Figure 2) that are present in the bile of man, rat, and dog27 were employed as guest molecules for complexation with HPβCD. Bile salts and their interaction with CDs are of interest as they bind competitively to drug:CD complexes in the small intestine, thereby releasing the drug.13,28,29 For this reason, it is important to understand how changes in the DS affect the strength of CD:BS complexes. Complexes between CDs and bile salts have already been studied (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.

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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,12Rdihydroxy-24-oxo-5β-cholan-24-yl]amino)ethanesulfonic acid), taurochenodeoxycholate (TCDC) (2-([3R,7R-dihydroxy-24-oxo5β-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-cholan24-oic acid N-(carboxymethyl)amide) and glycochenodeoxycholate (GCDC) (3R,7R-dihydroxy-24-oxo-5β-cholan-24-oic acid N-(carboxymethyl)amide) were purchased from Sigma-Aldrich (St. Louis, MO). Samples of HPβCDs having different average DS were purchased from Sigma-Aldrich and CycloLab (Budapest, Hungary). All other chemicals were of analytical grade or higher and milli-Q water was used for the solutions. All chemicals were used without further purification. Mass Spectroscopy. MALDI-TOF MS was performed using a fast evaporating nitro-cellulose (FENC) matrix on a Reflex III instrument (Bruker Daltonics, Bremen, Germany). A thin layer of freshly prepared saturated R-cyano-4-hydroxycinnamic acid (CCA) in acetone was deposited on a MALDI target plate and allowed to dry. The CD was then deposited directly to the MALDI target plate by adding a 1 μL droplet of a 1:1 solution of the CD (10 mM) and a 1:4 (v/v) mixture of nitro-cellulose and saturated CCA in an aqueous solution of 0.1% triflouroacetic acid and 80% acetonitrile. NMR Spectroscopy. The NMR experiments were carried out at 25 °C on a Bruker Avance-600 NMR (Bruker Biospin, Rheinstetten, Germany) spectrometer operating at 14.1 T and equipped with a cryogenically cooled probe: 32 scans were acquired for the 1D 1H experiments, 5120 scans were acquired for the attached proton test (APT) experiments, while for the twodimensional HSQC and HMBC experiments, 32 and 64 scans, respectively, were acquired for each of the 128 t1 increments. The CD samples were run at 10 mM in D2O. The ROESY spectra were recorded on samples containing 10 mM CD and 10 mM BS in D2O and 16 scans were acquired for each of the 256 t1 increments. (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.; Nun~ez, E. R.; Tato, J. V. Supramol. Chem. 2003, 15, 33–43. (40) Cabrer, P. R.; Alvarez-Parrilla, E.; Meijide, F.; Seijas, J. A.; Nun~ez, 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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Figure 2. Names and structure of the six investigated bile salt anions.

Isothermal Titration Calorimetry (ITC). Prior to preparing the solutions for the ITC experiments, the solid powders of the BSs and CDs were dehydrated in vacuum at 55 °C for at least 48 h. Immediately after dehydration, the appropriate amounts were weighed out and the solutions were prepared by dissolving the powders in a 50 mM phosphate buffer, pH 7.1. The titrations were performed on a Microcal VP-ITC titration microcalorimeter (MicroCal, Northhampton, MA) at 25.0 °C and atmospheric pressure. The syringe was loaded with the CD solution and titrated into the cell containing the bile salt solution. In all the titration runs the volume of the first injection was 2 μL followed by 27 injections of 10 μL, with at least 240 s between each injection. The data point corresponding to the 2 μL injection was deleted prior to the nonlinear regression described below. All titrations were performed in triplicate. 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 data were fitted to a model based on one set of identical, independent binding sites using Microcals ITC data analysis application for the Origin software package (version 7.0). The regression parameters are the stoichiometry, n, the molar enthalpy of complexation, ΔH°, and the stability constant, K. The stability constant and molar enthalpy enabled the calculation of the standard Gibbs free energy of binding (ΔG°) and the standard change in entropy (ΔS°) according to

Figure 3. Mass spectrum of HPβCD with DS = 0.63. The peak at m/z = 1216.5 corresponds to the Naþ adduct of HPβCD with only one 2-hydroxypropyl substituent per CD-ring, and the peak at m/z = 1681.8 corresponds to the Naþ adduct of HPβCD with nine substituents. Kþ adducts are also present in the spectrum. Table 1. Names and DS of the Studied HPβCDs As Determined by 1 H-NMR and MALDI-TOF MS

ΔG° ¼ - RT ln KS ¼ ΔH° - TΔS°

name

DS (1H NMR)

DS (MALDI-TOF MS)

where R 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 CD solutions were in the range of 1.25-20 mM and the BS solutions were in the range 0.125-2 mM. The higher the stability constants were, the more diluted were the solutions.

HP054 HP063 HP082 HP102 HP106

0.54 0.63 0.82 1.02 1.06

0.59 0.67 0.95 1.05 1.09

Results and discussion Characterization of Cyclodextrins. Using NMR and MALDI-TOF MS, the substituted cyclodextrins were characterized with respect to the degree of substitution and substitution pattern. From the MS spectra, of which an example is shown in Figure 3 and the rest are presented in the Supporting Information, it is seen that each of the purchased cyclodextrin samples are highly polydisperse. In all samples 6-9 HPβCDs of various DS are symmetrically Langmuir 2010, 26(23), 17949–17957

distributed around the average DS. From the peak intensities, the average degree of substitution can be estimated as P Ii DSi i P DS ¼ Ii i

where Ii denotes the intensity of the ith peak and DSi denotes the degree of substitution corresponding to the ith peak. The average DS of the used CD samples were also calculated from the 1H DOI: 10.1021/la103124n

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

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 obtained by NMR. Due to variations in the ionization and vaporization potentials among the differently substituted molecules in each sample, there might be a bias in the DS determined by MS. In the following the DS obtained by NMR will therefore be used. The substitution patterns were determined from both the 1D and 2D NMR experiments (all 1D 1H and 13C spectra as well as 17952 DOI: 10.1021/la103124n

representative 2D spectra are shown in the Supporting Information). Substitution at O2 gives rise to a downfield shift (∼0.2 ppm) of H1 and H3, as well as an upfield shift (∼2 ppm) of C1 and C3 and a downfield shift (∼8 ppm) of C2 (see Figure 1 for numbering of the CD nuclei).37,44 All of these extra peaks resulting from the partial substitution at O2 are observed in the (44) Tongiani, S.; Velde, D. V.; Ozeki, T.; Stella, V. J. J. Pharm. Sci. 2005, 94, 2380–2392.

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Figure 5. Partial ROESY spectrum of the complex of HP106 with GC. The most important intermolecular interactions between CD protons (H) and BS protons (P) are encircled.

NMR spectra, proving the existence of substitution at this oxygen. Furthermore, a three-bond coupling between the substituent and the proton on the substituted C2 appear in the HMBC-spectra (Figure 4), providing additional evidence of O2-substitution. The percentage of O2s that are substituted may be quantified from the peak areas of H1 and H10 (the C1 proton next to a substituted C2), as these peaks are well separated. Dividing this percentage by the DS yields the fraction of substituents bound to O2. This reveals that all of the HPβCDs have 50-60% of the substituents located at O2. It is not possible, however, to determine if the remaining 40-50% of the substituents are located on O3 or O6. The unsubstituted C6 appears as an easily recognizable negative peak in the APT spectra and substitution at O6 would definitely shift this peak significantly downfield (a shift of 8-10 ppm is expected as observed for C2), but such a peak does not appear in any of the spectra. Similarly, there are no features in the NMR-spectra to support substitution at O3. The HMBC spectra only show the above-mentioned coupling between Ca and H2sub as well as the expected three-bond coupling between Ca and the HP methyl protons, Hc, but no other couplings with Ca are observed. The expected peaks and couplings resulting from substitution at O3 and/or O6 are probably too broad and too weak to be observed. 2D ROESY NMR. A structural characterization of selected complexes was made using 2D ROESY NMR. Two weakly binding complexes (HP054 and HP106 with GC) and two strongly binding complexes (HP054 and HP106 with GCDC) were investigated. The assignments of the spectra were made from the HSQC and HMBC spectra of the complexes, and parts of the assignments are shown in the projections of the ROESY spectrum in Figure 5. All four ROESY spectra exhibit the same features: (i) The interior CD proton H5 interacts with the BS protons P21, P22 and P23; (ii) the interior CD protons H3 and H30 (the H3 next to a substituted C2) interact with the BS protons P15 and P18; (iii) the proton, Hb, on the hydroxypropyl side chain interacts with the BS proton P19; (iv) no interactions are observed between the exterior CD protons H2 and H4 and the BS protons. These features are similar to the observations made for other CD:BS complexes,37,39,41 Langmuir 2010, 26(23), 17949–17957

and suggest the formation of inclusion complexes in which parts of the BS conjugation tail and D-ring are included in the CD cavity. The interaction between Hb and P19 shows that the HP side chains may interact with the parts of the BS protruding from the CD cavity. Isothermal Titration Calorimetry. Figure 6 shows two examples of the integrated peak area data after subtraction of the heat of dilution. From the fit of the model to the data, the complexation stoichiometry, n, the molar enthalpy of complexation, ΔH°, and the stability constant, K, were obtained and were used to calculate ΔG° and ΔS°. The values of K, ΔG°, ΔH°, and ΔS° for all complexes are listed in Table 2. Complex Stoichiometries. The complex stoichiometries, n, yielded as a fitting parameter from the model fit, in most cases fell within the interval 0.9-1.1, suggesting a 1:1 binding mode. However, in some complexes formed with TCDC, n was consistently lower than 1, reaching values as low as 0.82 CDs per BS, and titrations involving the highest-substituted CDs (HP102 and HP106) yielded values of n up to 1.2. These deviations from n = 1 do not change the overall conclusion that they form 1:1 complexes, which is in line with previous investigations of CD:BS complexes,31-37,41 although it has also been reported that TDC and GDC form 2:1 CD:BS complexes with natural βCD30,39,40 and possibly also with modified βCDs.22 The formation of 2:1 CD:BS complexes in addition to the formation of 1:1 complexes cannot be ruled out, but the association constant for the binding of the second CD to the BS must be much smaller than for the binding of the first CD. Otherwise, the chosen model would not have 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 function of the average degree of substitution in Figure 7. Some of the trends have previously been observed. In the case of BSs, the most important structural feature is the position of hydroxyl groups. The BSs are thought to enter the CD cavity with the side chain first, and the presence of a hydroxyl group on C12 sterically hinders the BS from penetrating deep into the CD cavity.33,41 As a DOI: 10.1021/la103124n

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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 strongly binding complex and comes from a titration of 0.253 mM GCDC with 2.50 mM HP063. In the case of a weak complex, the enthalpogram looks like the one in panel B, where 1.003 mM TC is titrated with 17.85 mM HP106. The fitting parameters corresponding to the best fits (solid lines) 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 host

K (M-1)

ΔG° (kJ/mol)

ΔH° (kJ/mol)

ΔS° (J/mol/K)

TC

HP054 HP063 HP082 HP102 HP106

1932 ( 36 1512 ( 71 1043 ( 32 861 ( 48 564 ( 6

-18.76 ( 0.05 -18.15 ( 0.12 -17.23 ( 0.07 -16.75 ( 0.14 -15.70 ( 0.03

-13.09 ( 0.30 -11.73 ( 0.31 -6.62 ( 0.16 -3.50 ( 0.12 -4.01 ( 0.08

19.01 ( 1.16 21.51 ( 1.44 35.57 ( 0.78 44.44 ( 0.83 39.23 ( 0.36

GC

HP054 HP063 HP082 HP102 HP106

2141 ( 87 1713 ( 29 1229 ( 72 913 ( 56 669 ( 38

-19.01 ( 0.10 -18.46 ( 0.04 -17.63 ( 0.14 -16.89 ( 0.16 -16.12 ( 0.14

-13.56 ( 0.45 -12.15 ( 0.14 -6.77 ( 0.38 -3.93 ( 0.03 -4.28 ( 0.25

18.28 ( 1.85 21.16 ( 0.60 36.44 ( 1.69 43.47 ( 0.58 39.71 ( 1.29

TDC

HP054 HP063 HP082 HP102 HP106

4052 ( 174 3902 ( 101 3101 ( 14 2632 ( 21 2100 ( 84

-20.59 ( 0.11 -20.50 ( 0.06 -19.93 ( 0.01 -19.52 ( 0.02 -18.96 ( 0.10

-15.56 ( 0.12 -13.04 ( 0.20 -7.09 ( 0.03 -3.82 ( 0.01 -3.59 ( 0.01

16.87 ( 0.74 25.03 ( 0.48 43.07 ( 0.14 52.68 ( 0.08 51.54 ( 0.34

GDC

HP054 HP063 HP082 HP102 HP106

4326 ( 98 4099 ( 100 3284 ( 72 2916 ( 198 2405 ( 213

-20.75 ( 0.06 -20.62 ( 0.06 -20.07 ( 0.05 -19.77 ( 0.17 -19.29 ( 0.22

-16.27 ( 0.05 -13.61 ( 0.11 -7.76 ( 0.05 -4.33 ( 0.10 -4.01 ( 0.13

15.03 ( 0.15 23.51 ( 0.58 41.30 ( 0.21 51.78 ( 0.89 51.27 ( 1.16

TCDC

HP054 HP063 HP082 HP102 HP106

73117 ( 6446 56763 ( 4257 38123 ( 862 28580 ( 743 20027 ( 102

-27.76 ( 0.22 -27.13 ( 0.18 -26.15 ( 0.06 -25.43 ( 0.06 -24.55 ( 0.01

-21.88 ( 0.08 -19.49 ( 0.47 -15.21 ( 0.09 -12.57 ( 0.08 -12.02 ( 0.05

19.70 ( 1.00 25.62 ( 1.06 36.69 ( 0.49 43.16 ( 0.38 42.05 ( 0.20

GCDC

HP054 HP063 HP082 HP102 HP106

84657 ( 244 65768 ( 919 41935 ( 191 30703 ( 571 22617 ( 226

-28.13 ( 0.01 -27.50 ( 0.03 -26.38 ( 0.01 -25.61 ( 0.05 -24.85 ( 0.02

-22.40 ( 0.03 -20.36 ( 0.35 -16.13 ( 0.03 -13.41 ( 0.03 -12.83 ( 0.11

19.21 ( 0.10 23.96 ( 1.12 34.39 ( 0.13 40.94 ( 0.26 40.33 ( 0.39

guest

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Figure 7. Measured stability constants for complexation with TC (9), GC (0), TDC (b), GDC (O), TCDC (2), and GCDC (4) as a function of the average degree of substitution of the HPβCDs. Stability constants for the natural βCD are taken from Holm et al., 2009.41

consequence of no hydroxyl groups at C12, the stability constants for the complexes involving the BS anions TCDC and GCDC are much higher than for the other BS. Interestingly, the hydroxyl group on C7 also has a pronounced effect on the complex stabilities. If the 12-OH sterically hinders the BS from a deeper insertion into the CD cavity, one would not expect the 7-OH to play a role in complex formation, since this part of the BS is too far away from the CD. Despite this, the HP-substituted CDs are clearly able to discriminate between BS with and without 7-OH, as seen from the stability constants, which are several times larger when 7-OH is missing (TDC and GDC) than when 7-OH is present (TC and GC). This suggests that even though the 12-OH sets 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 the BS that are outside the CD cavity. For all investigated complexes, the type of conjugation of the BS side chain only has a minor effect on the complex stabilities such that the glycoconjugated BS form slightly more stable complexes than the corresponding tauroconjugated ones. This is in accordance with previous results.22,34,41 As seen from Figure 7, the stability constants decrease with increased DS in a similar way for all types of BS, with the exception that TDC and GDC bind better to the substituted CDs than to the natural βCD. The decrease in complex stability with increased HP-substitution has previously been observed for Langmuir 2010, 26(23), 17949–17957

Figure 8. ΔH°, TΔS°, and ΔG° for the complexation of GC (0), GDC (O) and GCDC (4) with HPβCDs of various degrees of substitution. Data for the natural βCD are taken from Holm et al., 2009.41 Data for the tauroconjugated BS are not plotted, but they are very similar to the glycoconjugated BS. All data are presented in Table 2.

complexation with a number of other guest molecules, including phenolphthalein19,20,45 and various drug molecules,17 but sometimes the complex stability also increases with increasing DS, as is the case for p-methyl red.19 The relation between complex stability and DS is discussed in the next section. Thermodynamics of Complexation. The enthalpy of complexation, ΔH°, is negative in all cases, but shows a strong dependence on the DS. As seen in Figure 8, ΔH° increases more or less linearly with increasing DS resulting in complexations that are close to being athermal in the case of the highly substituted (45) Rao., C. T.; Pitha, J.; Lindberg, B.; Lindberg, J. Carbohydr. Res. 1992, 223, 99–107.

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

CDs. This decrease in enthalpic contributions to the complex stabilities is largely compensated by the entropy of complexation, which increases with increasing DS in a similar fashion, as shown in Figure 8. As a result, the free energy of complexation varies relatively little as a function of the DS (Figure 8). Whereas, ΔH° for the complexation with GC ranges from -24.5 to -4.3 kJ/mol, ΔG° only changes from -19.5 to -16.1 kJ/mol when going from the natural β-CD to DS ≈ 1. This significant enthalpy-entropy compensation is illustrated in Figure 9. Enthalpy-entropy compensation has previously been observed for a large variety of CD inclusion complexes14,18,25 including CD:BS complexes.22,41 Though the phenomenon of enthalpy-entropy compensation has been reported a number of times, the molecular (“extra-thermodynamic”) mechanism behind this relation is generally highly controversial and has been argued to be based upon artifacts.46-52 A discussion of this, however, is beyond the scope of the present work. The effects of side chains substituted onto the rim of the CD has previously been reported to lead to larger ΔH° and ΔS° values in the case of the association of larger polycyclic guests with sulfobutylether-βCD18 and for the complex formation between monocarboxylic acid and HPβCD.25 That is in line with the results presented here. The observations that an increased number of side chains led to an increase in ΔS° were thought to be caused by the additional hydrophobic interactions between the side chains and the hydrophobic guest molecules.18,25 This interpretation may also explain the variations in ΔH° and ΔS° with the DS presented here. One might expect that an extended hydrophobic cavity would increase the complex stability, as observed in the case of methylated R- and βCDs,12,53 but the stability constants clearly decrease with increased substitution. Steric effects arising from the HPchains partially blocking the cavity entrance could be an impor(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.

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tant factor, but the HP-chains might also alter the rigidity and structure of the CD and thereby result in a less optimal fit between the CD and the guest molecule. Yong et al.23 have carried out molecular dynamics simulations of the structural behavior of natural βCD and differently substituted HPβCDs in water. Side chains at O2 induce a more “spread out” conformation of the CD, where the wide cavity opening is wider and the narrow opening is narrower. This may lead to a less tight fit between the bile salt and the CD, resulting in lower binding stability, a less favorable ΔH° because of weaker attractive van der Waals interactions, and possibly a more favorable ΔS° due to a looser fit and increased number of conformations of the complex. These thermodynamic consequences are in accordance with the experimental observations. The simulations also suggest the formation of intramolecular hydrogen bonds between the hydroxyl group of the substituent and the hydroxyl group at C3. If these are disrupted upon complexation, a less favorable ΔH° and a lower complex stability is expected. It is noteworthy that the enthalpy and entropy of complexation change linearly with the DS, as seen in Figure 8. That means that the effects of the HP-chains are additive in the sense that each of the added HP-chains contributes equally to the increase in ΔH°. One may view the ΔH° for the complexation with HPβCD as stemming from ΔH° for the complexation with natural βCD plus ΔH° 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, one may conclude that each HP-substituent adds 3.6 kJ/mol to the enthalpy of complexation and also 3.6 kJ/mol to TΔS° of complexation. In the cases of GC and GCDC, the contributions to ΔH° are 2.8 and 2.2 kJ/mol, respectively, and the contributions to TΔS° are 2.4 and 1.7 kJ/mol, respectively. If the increase in TΔS° mainly stems from the increased release of ordered water molecules, the surface area that each HP-chain dehydrates may be calculated. Costas et al.54 have calculated that the rearrangement of water molecules near a hydrophobic surface contributes to TΔS° with -38.3 mJ per m2 of solute surface area at 298 K. Using the above increment in TΔS° (2.4 kJ/mol) for each HP chain interacting with GC yields a dehydrated surface area of 10.4 A˚2 per HP chain or 2.7 ordered water molecules released for every 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 the increased release of ordered water molecules is the main contributor 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 clearly distinguishable groups. All of the complexes involving TCDC or GCDC lie on a straight line in the plot, while the other complexes involving the remaining four bile salts form a second group. This second group might be separated into two groups: one group for the TDC and GDC bile salts and another group for the TC and GC bile salts. The linear correlation of TΔS° with ΔH° within a series of complexes suggests that the overall binding mode is the same for all the complexes in that series. Thus, the binding mode of TCDC and GCDC to HPβCD is different than the binding mode of TDC/GDC/TC/GC to HPβCD. This conclusion is in line with the observation that TCDC and GCDC penetrate deeper into the CD cavity than the other bile salts. Interestingly, the number of HP-chains does not change the overall binding mode. It is the structure of the bile salt molecule (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 overall binding mode. The slope (R) and intercept (TΔS0) of the enthalpy-entropy compensation plot has previously been interpreted as measures of the degree of conformational changes and the degree of desolvation, respectively.14 A slope close to 1 means that the complex possesses considerable flexibility and can undergo significant conformational changes upon complexation, whereas the intercept reflects the entropic contribution to complex stability when there is no enthalpic contribution and is related to the degree of desolvation of the host and guest molecules upon complexation. From the enthalpy-entropy compensation plot, it is evident that the 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/GCDC bile salts into the CD cavity and the associated larger extent of dehydration. For comparison, the analysis of more than 1000 different complexes with natural CDs and more than 100 modified CDs yielded intercepts of 12 and 17 kJ/mol, respectively.14 It is clear that the interaction between HPβCD and TCDC/GCDC bile salts results in an unusually large value of TΔS0 suggesting that dehydration plays an exceptionally large role in these complexes.

calorimetry for complexes between six bile salts and hydroxypropylated β-cyclodextrins with varying degrees of substitution. The enthalpy of complexation increases strongly and linearly with the degree of substitution, but this is largely compensated by a similar increase in the entropy of complexation, so that the standard free energy of complexation increases slightly with the degree of cyclodextrin substitution. The overall complex structure and stability is determined by the structure of the bile salt and in particular the presence or absence of the C-12 hydroxyl group, as previously described for natural β-cyclodextrin.30,41 The hydroxypropyl chains on the rim of the cyclodextrin are thought to align themselves with the hydrophobic parts of the bile salt molecule that protrudes from the cyclodextrin cavity. This results in a release of ordered water molecules, which is responsible for the significant variations in the enthalpy and entropy of complexation, but has a somewhat smaller effect on the complex stability. It is estimated that each hydroxypropyl chain dehydrates a surface area of around 10 A˚2. Since the hydroxypropyl chains have the same qualitative effect on the complexation with all six different bile salts, one may expect a similar effect on complexation with other guest molecules.

Conclusion

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

Stability constants and the associated enthalpies and entropies of complexation were determined by isothermal titration

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