Hydration Differences Explain the Large Variations in the

Article pubs.acs.org/JPCB

Hydration Differences Explain the Large Variations in the Complexation Thermodynamics of Modified γ‑Cyclodextrins with Bile Salts Jonatan Køhler,†,‡ Christian Schönbeck,†,‡ Peter Westh,‡ and René Holm*,† †

Pharmaceutical Science and CMC Biologics, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark NSM, Research Unit for Functional Biomaterials, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark

‡

S Supporting Information *

ABSTRACT: The structure and thermodynamics of inclusion complexes of seven different γ-cyclodextrins (γCDs) and three biologically relevant bile salts (BS) were investigated in the present study. Natural γCD and six modified γCDs [two methyl-γCDs, one sulfobutyl ether-γCD (SBEγCD), and three 2hydroxypropyl-γCDs (HPγCD)] and their complexes with BS were investigated by isothermal titration calorimetry, NMR, and molecular dynamics simulations. With the exception of the fully methylated γCD, which did not bind the BSs investigated, all of the γCDs formed 1:1 complexes with the BS, and the structures were similar to those with natural γCD; i.e., the modifications of the γCD had limited structural impact on the formation of complexes. Isothermal titration calorimetry was carried out over in the temperature interval 5−55 °C to enable the calculation of the stability constant (K) and the thermodynamic parameters enthalpy (ΔH°), entropy (ΔS°), and heat capacity (ΔCp°). The stability constants decreased with an increased degree of substitution (DS), with methyl substituents having a lower effect on the stability constant than the sulfobutyl ether and hydroxypropyl substituents on the stability constants. Enthalpy−entropy compensation was observed, since both enthalpy and entropy increased with the degree of substitution, which may reflect dehydration of the hydrophobic surface on both CD and BS. Calculations based on ΔCp° data suggested that each of the substituents dehydrated 10−20 (hydroxypropyl), 22−33 (sulfobutyl ether), and 10−15 Å2 (methyl) of the BS surface area, in reasonable agreement with estimates from the molecular dynamics simulations. Combined with earlier investigations on modified βCDs, these results indicate general trends of the substituents on the thermodynamics of complex formation.

1. INTRODUCTION Cyclodextrins (CDs) are cyclic oligosaccharides consisting of glycopyranose units linked by α-1,4-glycosidic bonds. The most commonly used CDs consist of six, seven, or eight units of glycopyranose, called α-, β-, or γCD, respectively. Limited rotation of the bonds within the CD structure makes CDs take the shape of truncated cones (Figure 1). The CD cone has a hydrophilic exterior and a hydrophobic interior cavity that can contain molecules of the right shape and size. This gives CDs the ability to make inclusion complexes with hydrophobic guest molecules in aqueous solution, a property that is widely used in several different disciplines, including pharmacy, to increase the apparent water solubility of poorly soluble drugs for oral drug delivery.1−4 Natural CDs can be modified by substituting the hydroxyl groups at O2, O3, and O6 with, for example, methyl or 2hydroxypropyl, resulting in changed physiochemical properties of the CD, for instance, increased aqueous solubility. So much effort has been put into designing substituted CDs with specific properties useful in different situations, that many different substituted CDs are commercially available. Detailed knowl© 2016 American Chemical Society

edge of the effects of the different substituents and the pattern of substitution is important in choosing the optimal CD for a given purpose. The interaction between βCDs and bile salts (BS) has been extensively described in the literature.5−10 The interaction between BSs and γCDs is fundamentally interesting from a physical chemical perspective and provides an excellent model system to investigate the potential effects of the substituents on γCDs and to compare them with substituted βCDs. The burial, or dehydration, of the hydrophobic surface in aqueous solution affects the observed thermodynamics of the interaction. In particular, the change in heat capacity, (ΔCp) seems to scale with the buried surface area, a relation that has also been observed in other systems, for example, the dissolution of hydrocarbons in water (Gill and Wadsö, 1976) and protein unfolding,11 so the phenomenon is of general importance. Studies of variously modified CDs, i.e., 2hydroxypropyl-β-CD (HPβCD) and methyl-β-CD (mβCD), Received: October 27, 2015 Revised: December 28, 2015 Published: January 5, 2016 396

DOI: 10.1021/acs.jpcb.5b10536 J. Phys. Chem. B 2016, 120, 396−405

Article

The Journal of Physical Chemistry B

Water used during the experiments was from a Millipore purification system (Billerica, MA). Sodium phosphate and D2O (99.9%) were obtained from Sigma-Aldrich. All other chemicals were of analytical grade and all chemicals were used without further purification. 2.2. Mass Spectrometry. To determine the average degree of CD substitution MALDI-TOF MS was performed as previously described previously,18 using a fast evaporating nitrocellulose (FENC) matrix in a Reflex III (Bruker Daltonics, Bremen, Germany). A thin layer of saturated α-cyano-4hydroxycinnamic acid (CCA) in acetone was deposited on the MALDI target plate and allowed to dry. The modified γCDs were then deposited directly onto the MALDI target plate by adding a 1 μL of a 1:1 solution of the modified γCD (1 mM) and a 1:4 (v/v) mixture of nitrocellulose and saturated CCA in an aqueous solution of 0.1% trifluoroacetic acid and 80% acetonitrile. 2.3. NMR Spectroscopy. All NMR experiments were carried out at room temperature on a Bruker Avance-600 NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) operating at 14.1 T and equipped with a cryogenically cooled probe. To characterize the CDs, 1H, 13C (APT), HMBC, and HSQC spectra were recorded. The CDs were dissolved at 10 mM in D2O. For the description and structural analysis of the complexes, HSQC and 2D 1H-ROESY experiments were recorded in 5 mM CD/BS in D2O. To assign the cross correlation peaks in the ROESY spectra, it was necessary to know the assignment of each proton in the 1H NMR spectra of the complexes. For these assignments HSQC spectra of the complexes were also recorded. The assignments of the HSQC spectra were based on the 1H and 13C assignments of the free CDs and BS. For the CDs, these were made from 1H NMR, 13 C APT, and HSQC spectra, while the assignments of the free BSs were obtained from the literature.18−20 2.4. Molecular Dynamics Simulations. Molecular dynamics simulations of free and complexed species were carried out using the NAMD code21 (version 2.10 for Linux multicore) in TIP3P water boxes with periodic boundary conditions. Pressure and temperature were maintained at 1.013 bar and 298 K, respectively. The side lengths of the cubic boxes were sufficiently large to avoid nonbonded interactions (cutoff at 12 Å) between mirror images of the solvated species. The side lengths thus ranged from 34 Å for the free BSs to 44 Å for the complexes with SBE-γCD. The natural γCD initial structure was obtained from the Cambridge Structural Database (CCDC 1126611). To generate the initial structures of the modified γCDs, substituents in random conformations were attached to the natural γCD structure. For the complexes, the nitrogen atom of the equilibrated BS structures were placed at the center of mass of the glycosidic oxygens of the equilibrated CD structures and orientated so that the steroid body protruded from the secondary CD rim. This orientation of the BS, in which the conjugation tail protrudes from the primary rim, was in accordance with the presently reported ROESY spectra and findings in the literature.1,17 Water boxes, which included sufficient sodium ions to neutralize the negative charges on BS and SBE substituents, were generated around the molecules using the “solvate” and “autoionize” functions in the VMD software.22 The CHARMM carbohydrate force field23,24 was used for the natural γCD and for the CD moiety of the modified CDs. Parameters for the CD substituents were automatically

Figure 1. Structures and names of the investigated γCDs and substituents. The labeling of the nuclei is also shown. The schematic structure in (B) covers all γCDs whereas the cone shape shown in (A) is for natural γCD.

complexed with BS showed that the values of ΔCp, and also ΔS° and ΔH° were largely determined by differences in buried nonpolar surface area (ΔASAnon).8,9,12−14 These observations suggest that the large variations in ΔS°, ΔH°, and ΔCp, observed for the complexation of BS with various βCDs, were primarily due to differences in buried surface area. Increased burial of hydrophobic surface leads to significant increases in enthalpy and entropy and to more negative values of the heat capacity. For the complexes with HPβCDs, this resulted in linear correlations between the degree of CD substitution and these three thermodynamic functions,12,13 but for the mβCDs, the effect of the substituents was more complex due to the larger range in their degree of substitution (DS).. These observations were supported both experimentally and by molecular dynamics (MD) simulations. While both natural and modified βCDs have been extensively investigated for their interaction with BS, much less is published on the larger γCD.7,15−17 Compared with βCDs, the natural γCDs interact structurally differently with BS, which are able to fit deeper into the larger cavity of γCD. The interaction between modified γCDs and BS has not been investigated; hence, the purpose of the current study was to conduct a comparative analysis on the interaction between the most common modified γCDs and three relevant BSs in order to assess the structural and thermodynamic effects of the various γCD modifications.

2. EXPERIMENTAL SECTION 2.1. Materials. Sodium salts of the bile acids, glycocholate (GC), glycodeoxycholate (GDC), and glycochenodeoxycholate (GCDC) were acquired from Sigma-Aldrich (St. Louis, MO) and their chemical structures are shown in the Supporting Information. Samples of γCD and 2-hydroxypropyl-γCD (HPγCD) with an average degree DS of 0.6 were purchased from Sigma-Aldrich. HP-γCD samples with reported DS 0.16 and 0.53 and methyl-γCD (Me-γCD) samples with reported DS 1.58 and 3.0 were purchased from CycloLab (Budapest, Hungary). The sulfobutyl ether-γCD (SBE-γCD) sample was kindly donated by Ligand Pharmaceuticals (La Jolla, CA). 397


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Table 1. Degrees of Substitution (DS) and Pattern of Substitution for all the Investigated CD Samples Measured by Mass Spectrometry (MS) or NMR CD (name)

DS (MS)

DS (1H NMR)

DS(O2), %

DS(O3), %

DS(O6), %

γCD HP016-γCD HP054-γCD HP060-γCD Me135-γCD Me300-γCD SBE057-γCD

− 0.18 0.53 0.65 1.6 − 0.37

0 0.16 0.54 0.60 1.35 3.00 0.57

0 51.2 56.6 60.6 41.4 (39.1)b 33.3 49.5

0 − − − 8.7 (27.7)b 33.3 40.3a

0 − − − 49.9 (33.1)b 33.3 10.2

a

This value could not be determined directly by any peak area, so it was calculated subtracting DS(O2) and DS(O6) from 100%. bThe peak areas of C4 and C4′ are used to determine DS(O3) instead of using the areas of the methyl carbons.

generated by the CGenFF program version 1.0.025,26 using CHARMM General Force Field version 3.0.1.27 All parameters for the BS were available from CHARMM General Force Field version 3.0.1.27 The simulations were run with 2 fs time steps. The lengths of the simulations varied as some systems required longer simulations to ensure good sampling. Simulations of the conformationally simple BSs were run for 10 ns, most free CDs and their complexes for 20 ns, and the free Me-γCDs were simulated for 30 ns due to their greater flexibility. In the subsequent analysis the initial equilibration periods, which were excluded from the sampling, lasted for 2 ns for the free BSs and 5 ns for free CDs and the complexes. The solvent accessible surface area (ASA) was calculated with the “sasa” function in the VMD software22 using a probe radius of 1.4 Å. The total ASA was divided into polar and nonpolar ASA by considering carbon and attached hydrogen to be nonpolar, while oxygen, nitrogen, and attached hydrogen were considered to be polar. 2.5. Isothermal Titration Calorimetry (ITC). All samples of BSs and CDs were dehydrated in a vacuum oven at 55 °C for at least 48 h prior to generation of the solutions for the ITC measurements. Dehydrated CDs and BS were weighed and dissolved in 50 mM phosphate buffer pH 7.0 to make solutions of approximately 10 mM and 1 mM of CDs and BSs, respectively. The concentrations of the substituted CDs were calculated based on the average molecular weights obtained by NMR. The low concentration of the BSs was chosen to ensure that the measurements were conducted below their critical micelle concentration (cmc),16,28−31 thereby avoiding enthalpy changes from micellation/demicellation as factors. The thermodynamic experiments were carried out using a Microcal VP-ITC microcalorimeter (MicroCal, Northhampton, MA) in a thermostated room at atmospheric pressure. All samples were degassed in a ThermoVac (MicroCal, Northhampton, MA) before the experiment. The titrations were performed by titrating the CD solutions into the BS solutions in the titration cell. The first CD injection was 2 μL and the subsequent 27 injections were set to 10 μL, with 300 s between each injection. The first injection was discharged from the data analysis. Measurements were performed at 10 °C intervals from 5 to 55 °C for each CB/BS combination. Heats of dilutions were measured separately for each CD and temperature by titrating the CD solutions into the phosphate buffer solution. These heats of dilution were subtracted from the results of the corresponding CD/BS titrations, yielding the heats of complexation. The results from the measurements were analyzed using Microcal’s ITC data analysis application for the Origin software package (version 7.0). A single set of identical and independent

binding sites was assumed, which yielded the following parameters when the data was fitted to the model: stoichiometry (n), the molar enthalpy of complexation (ΔH°, assuming ideal dilute solutions and hence that the measured ΔH was equal to ΔH°), and the stability constant (K). The standard Gibbs free energy of complexation (ΔG°) and the standard change in entropy (ΔS°) were calculated from ΔH° and K, in accordance with ΔG° = −RT ln(K ) = ΔH ° − T ΔS°

(1)

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

3. RESULTS AND DISCUSSION In the present study inclusion complexes between seven γCDs (natural γCD, HP-γCDs, Me-γCDs, and SBE-γCD) and the three BSs found in the human intestine were investigated using structural and thermodynamic analysis. NMR and mass spectrometry were used to characterize the different CDs, and 2D-NMR techniques and MD simulations were used to analyze the structure of the complexes. The thermodynamics of the interaction was investigated by ITC - all aimed at obtaining a systematic evaluation of how differences in substitution of γCDs affect the structure and thermodynamics of CD/BS complexes. 3.1. Characterization of Cyclodextrins. The natural γCD and the fully methylated Me-γCD were monodisperse, whereas all other CDs were found to be polydisperse by MALDI-TOF (MALDI-TOF spectra can be found in the Supporting Information). The patterns and degrees of substitution were investigated by 1H NMR and 13C NMR (see Supporting Information) and are presented in Table 1. The 1H spectra of the substituted γCDs showed clearly distinguishable peaks from H1 (CD proton 1) and H1′ (=H1 when there was another substitution at O2). The degree of substitution at O2 (DS(O2)) was calculated from the relative peak areas. Of the three HP-substituted CDs, only DS(O2) was seen as a distinct substitution, whereas both the 1H and 13C peaks from the substituted H3/C3 and H6/C6 were either superimposed on other peaks, or were too broad or too weak to determine. A similar problem has previously been reported for HP-βCDs,8 where only the fraction at DS(O2) was reported. DS(O2) constituted 50−60% of the total DS for all three investigated HP-γCDs, indicating that O2 was the primary substitution site. Several published of HP-βCDs have revealed that slightly more than half of the substituents were located at O2,32 while the rest were distributed equally between O3 and O6, or 2:1 between O3 and O6.33 As argued by Tongiani et al.,34 the amount of chemically equivalent carbons such as C6 and C6sub (=C6 when another 398


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Figure 2. Assigned partial ROESY spectrum of the HP060-γCD/GCDC complex. CD protons are denoted by “H” and BS protons by “P”.

Table 2. Distribution of Substituents in the Cyclodextrins (CD)s Built for the Molecular Dynamics Simulations experimental CD

simulated CD

DS

γCD mγ135

gCD mg138a mg138b HPg063 SBg063

0 1.38 1.38 0.63 0.63

HPγ060 SBγ057

O2 substitution at glucose units no. − 1, 1, 1, 1,

2, 4, 6, 7 (45%) 2, 4, 6, 7 (45%) 3, 6 (60%) 6 (40%)

O3 substitution at glucose units no.

O6 substitution at glucose units no.

− 6 (10%) 1, 3, 6 (27%) 8 (20%) 3, 5 (40%)

− 1, 3, 4, 6, 8 (45%) 1, 4, 8 (27%) 5 (20%) 1 (20%)

Supporting Information. The spectra were generally difficult to analyze due to the large extent of overlapping peaks. All complexes had relatively similar structures. The most likely structure was similar to that reported for natural γCD-BS complexes,17 in which the BS has entered the cavity of the CD from the secondary opening and the side chain protrudes from the primary rim. The cavity of γCD seemed to included most of the steroid body of the bile salts with the C-ring, D-ring, and part of the B-ring included, i.e., the substitution on the CDs had a limited effect on the physical position of the BSs within the CDs. 3.3. Molecular Dynamics Simulations. Three BSs (GC, GDC and GCDC), 5 CDs (natural γCD, one HP-γCD, one SBE-γCD, and two Me-γCDs) and the resulting 15 complexes were simulated. The exact substitution patterns of the CDs are shown in Table 2 and are based on the NMR analysis. Simulations of the free BSs revealed an overall rigid overall structure, in which only the chain on the D-ring underwent significant conformational changes. Minor conformational

substitution was present) or the methyl groups of the substituents may be compared using the 13C peak areas. DS(O3) and DS(O6) were calculated for the SBE057-γCD and Me135-γCD samples using this approach. Some ambiguity existed regarding the exact distribution between O3 and O6 for Me135-γCD. Using the 13C peak areas of the methyl groups indicated that 9% and 50% of the methyl substituents were located at O3 and O6, respectively. Because of some overlap of the O3 methyl peak with the negative peak of C6 in the APT spectrum the value of DS(O3) is underestimated. Alternatively, the area of C4 can be compared to the area of C4′ (C4 where the neighboring O3 is substituted) resulting in 28% and 33% methyl substituents on O3 and O6, respectively. As can be seen from the data in Table 1, the average degree of substitution measured by MS and NMR was in the same range, confirming the findings. 3.2. Structural Characterization of Complexes by ROESY NMR. A representative ROESY spectrum is shown in Figure 2. The complete analysis of the 2D spectra is in the 399


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Table 3. Stability Constants (K), Enthalpy (ΔH°), Entropy (ΔS°), and Gibbs Free Energy (ΔG°) for Complexation for All Investigated CD-BS Complexes Obtained at 45°Ca

a

host

guest

γCD γCD γCD SBE057-γCD SBE057-γCD SBE057-γCD Me135-γCD Me135-γCD Me135-γCD HP016-γCD HP016-γCD HP016-γCD HP054-γCD HP054-γCD HP054-γCD HP060-γCD HP060-γCD HP060-γCD

GC GCDC GDC GC GCDC GDC GC GCDC GDC GC GCDC GDC GC GCDC GDC GC GCDC GDC

K (M−1) 3510 56200 11300 1210 25500 7250 1360 25700 7890 2990 50800 12900 2020 32400 8300 1380 21600 5550

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

ΔH° (kJ/mol) −20.0 −21.2 −15.4 −15.1 −19.0 −13.5 −6.7 −10.9 −12.0 −15.9 −20.0 −14.9 −10.9 −16.0 −10.6 −9.7 −12.7 −7.8

41 861 472 53 528 144 106 941 409 117 1200 486 79 666 162 42 813 219

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.0 0.1 0.5 0.1 0.1 0.4 0.1 0.2 0.3 0.1 0.1 0.2 0.0 0.1 0.2 0.1 0.1

ΔS° (J/mol/K)

ΔG° (kJ/mol)

5.0 24.4 29.3 11.5 24.7 31.5 39.1 50.2 38.1 16.6 27.2 31.9 29.0 36.1 41.9 29.5 43.1 47.3

−21.6 −28.9 −24.7 −18.8 −26.8 −23.5 −19.1 −26.9 −24.5 −21.2 −28.7 −25.0 −20.1 −27.5 −23.9 −19.1 −26.4 −22.8

The uncertainties given are the standard error of the nonlinear regression obtained by the iterative data fitting procedure.

independent binding sites yielded a good fit to the data and was therefore applied. The stoichiometry was 0.8 to 1.1 for the vast majority of complexes, suggesting 1:1 complexes in accordance with previous reports with γCD/BS complexes.10,17,36,37 A few complexes had values of n < 0.8 at certain temperatures. These titrations were discarded as the model of one set of identical and independent binding sites did not describe the data in those instances. 3.5. Stability Constants. The stability constants obtained at 45 °C are presented in Table 3 and the rest are in the Supporting Information, together with the van’t Hoff plots. The stability constants varied as a function of CD, BS, and temperature, however, the fully methylated γCD showed no detectable binding regardless of BS and temperature. The most important factors for the stability constant were the number and positions of OH-groups on the BSs. The affinities for BSs consistently decreased in the order GCDC > GDC > GC, as previously observed for natural γ-CD.15 The complexes of GC and GDC were destabilized by the unfavorable inclusion of the OH group at C12, while the presence of an additional OH group at C7 of GC resulted in the lowest BS affinity for the CDs. Among the CDs, the strongest complexes were formed with the natural γCD. The stability constants for the natural γCD complexes were in the same range as previously reported, thus validating the findings.7,15,17,36 When compared to βCD complexes of similar DS, the γCD complexes were generally found to form stronger complexes with GC and GDC, and weaker complexes with GCDC.5,8,9,14 The influence of the DS was tested for the HP substituents and the results are shown in Figure 3. Four different HP-γCDs with a DS of 0−0.6 (including the natural γCD as DS = 0) were investigated in the present study. The trend from Figure 3 clearly demonstrates that the stability constants decreased with increasing DS, in agreement with results for HP-βCDs,8 which was argued to be a consequence of partial blocking of the cavity by the substituents and/or of an altered rigidity and structure of the CD leading to a less optimal fit.8 This explanation could also apply to the results from the present study.

changes were also observed for the free γCD, in which the circular ring structure was retained throughout the simulation. The modified CDs, however, exhibited a somewhat larger degree of flexibility, with increased distortion of the circular macrocycle. For HP-γCD and SBE-γCD, this was manifested in small and transient tilts of the glucose moieties around the glycosidic bonds; but for the highly substituted Me-γCDs rotations of single glucose moieties were observed, so that the primary hydroxyls appeared at the secondary rim of the CD and vice versa. Such rotations were accompanied by strong distortions of the overall circular structure and lasted for several ns. Similar observations were previously made for methylated βCDs13 and probably result from disruption of the hydrogen bond network that is thought to stabilize the circular structure of the natural CDs.35 Rotations and distortions appeared much more frequently for Me-γCD with most substituents on O6 (mg138b), than when on O3 (mg138a), indicating that methylation at O3 destabilize the circular CD structure more than methylation at O6. Initially, the BSs were only marginally included in the CDs with the glycine nitrogen located in the middle of the CD. Within the first ns, the CDs moved to a stable position on the steroid body of the BS, which agrees well with the experimentally observed NMR ROESY interactions. The CDs moved somewhat back and forth on the steroid body and in turn included most parts of the steroid body, with the exception of the outermost parts of the A-ring. There seemed to be no significant structural differences between the various complexes, either with respect to the type of CD or the type of BS. The MD simulations thus support the conclusions from the ROESY spectra. 3.4. Isothermal Titration Calorimetry. Titrations were conducted for all combinations of CDs and BSs at temperatures 5−55 °C at 10 °C intervals. Representative examples of the obtained enthalpograms are be found in the Supporting Information. Some of the complexes had athermal reactions at temperatures about 15−25 °C, and could not be studied calorimetrically in this range. For the majority of the measurements, a model based on one set of identical and 400


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Figure 4. ΔH° and TΔS° values plotted as a function of DS for HPγCD complexes. The ΔH° and TΔS° values are represented at 25 °C, based upon extrapolation of measured data at 5−55 °C. The different symbols refer to complexes with GC (■), GDC (●), and GCDC (▲).

ΔS° than HP-γCDs with a similar DS, showing that SBE side chains affect the thermodynamics of complexation to a lesser degree than the HP side chains. By assuming linear dependence of DS, values of ΔH° and ΔS° were extrapolated to a lower DS based upon the Me135-γCD complexes, based on assumptions from Me-βCDs data.13 At comparable DS (0.55), Me-γCD had lower values of ΔH° and ΔS° than HP-γCDs and SBE-γCDs, except for the SBE057-γCD-GCDC complex, indicating that methyl substituents had the smallest effect on the thermodynamics of complexation. Given that these conclusions are drawn from extrapolations, a wider variety of SBE-γCDs and Me-γCDs are needed to confirm them. The precise values of ΔH° and ΔS° depended on the type of side chain and BS, but a high ΔH° was largely compensated by a high TΔS° for all CDs; hence the difference in ΔG° was small. This enthalpy−entropy compensation, which is illustrated in Figure 5, has been reported for several times for CD/ BS systems,5,18 but no definitive explanation exists. All complexes with the same type of BS follow the same straight line in Figure 5 regardless of CD, which suggests that all substituents have the same overall effect on enthalpy and entropy. Schönbeck et al.9 observed a similar behavior for βCD complexes and suggested that the common denominator was the dehydration of those parts of the hydrophobic guest molecules that protruded from the CD cavity, which led to an increase in both ΔH° and ΔS°. The amount of dehydration depended on the number and size of the side chains, which might explain the increase in ΔH° and ΔS° with an increasing DS seen in Figure 4. This would also explain why the larger HP side chains caused higher ΔH° and ΔS° values than the smaller methyl side chains relative to their respective DSs. A linear correlation of ΔH° and ΔS° has been observed for βCD-BS complexes and can be interpreted as the members of the same BS group binding to the CD via a common binding mechanism.17 Thus, Figure 5 suggests that all three BS have a different binding mechanisms with γCDs, which was not apparent from the structural analysis. This was also in contrast to what has been found for various βCD-BS complexes and natural γCD-BS complexes for which two groups are observed,

Figure 3. Stability constants of HP-γCD complexes as a function of DS at 45 °C. The different symbols refer to complexes with GC (■), GDC (●), and GCDC (▲).

The DS of the SBE-γCD was comparable with two of the HP-γCDs used and led to similar stability constants. SBE-βCD (DS = 0.91) complexes have previously been reported to form more stable complexes with BSs than HP-βCD complexes of similar DS, but comparable in strength to less substituted (DS = 0.54) HP-βCD complexes.14 This relative difference was probably a reflection of the difference in size between β- and γCDs. In the case of SBE-βCD, the long and flexible substitution is charged in the end and can thereby be place far from the hydrophobic bile salt. HP-βCD have a hydrophobic hydroxyl group in the second position of the substitution, why it may be more difficult to avoid the contact with the bile salt if the degree of substitution is high as it induces a range of steric hindrances within the HP-βCD molecule. In contrast to this, the bile salts are positions different in the γCDs. Further, due to the larger size the possibility of positioning the hydroxyl group from the substitution on HPγCD without being in contact with the bile salt appears easier, i.e., the similar stability constant relative to SBE-γCD. Me135γCD (DS = 1.35) complex stabilities were similar to those of the HP060-γCD (DS = 0.60) complexes. This suggests that, compared to the sulfobutyl ether or hydroxypropyl substituents, methyl substituents in γCDs had a less negative effect on the stability constants, when compared to the sulfobutyl ether or hydroxypropyl substituents. 3.6. Thermodynamics of Complexation. The entropies and enthalpies decreased with increasing temperature for all complexes. Both enthalpies and entropies were affected by the type and DS of the substituent on the γCD. Side chains on βCDs increase both the ΔH° and ΔS° of complexation,8 which was also observed in the present work. The ΔH° and ΔS° of HP-γCDs showed a linear dependence on DS (Figure 4), which was similar to the trend reported for HP-βCDs.8 At 25 °C, the SBE-γCD (DS = 0.57) complex had lower values of ΔH° and 401


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was very poor and no reliable result was obtained. The results show that SBE substituents had the greatest effect on ΔCp° followed by HP substituents and finally the methyl substituents, which have the smallest effect on ΔCp°. These results reflect the relative sizes of the substituents and agree with results for HP and methyl substituents on βCDs.12,13 There was a clear division between the ΔCp° of the dihydroxy BS complexes and the trihydroxy BS complexes in all cases, except for SBE057-γCD. GC complexes had substantially lower negative values of ΔCp°. Compared to GDC and GCDC, the extra hydroxyl group on GC makes the hydrophobic surface area available for dehydration smaller than for GDC and GCDC. SBE is larger than the other substituents and may be able to reach hydrophobic surface areas on GC that are inaccessible to the other substituents. The ΔCp° values reported for βCD-GDC complexes were generally less negative than for similar γCD complexes, whereas ΔCp° values for βCDGCDC complexes were more negative than for similar γCD complexes.12−14 This indicates structural differences between βCD and γCD complexes and supports the conclusions from the structural analysis. The change in nonpolar solvent accessible surface area (ΔASAnon) can be further quantified. Schönbeck et al.13 obtained experimental values for the relationship between ΔCp° and ΔASAnon and found an average value (±SD) of 1.8 ± 0.4 J/mol/K/Å2. Combining this value with the dependences of ΔCp° on DS (Table 5), the ΔASAnon contribution from each

Figure 5. Entropy-enthalpy compensation plot for all investigated complexes. Regressions have been carried out for groups of identical BS (GC = blue, GDC = red, and GCDC = black) to show the linearity within the groups. The natural γCD complexes are marked, and modified γCD complexes have different symbols (▲ = HP-γCD, × = SBE-γCD, and Δ = Me-γCD).

one for GC and GDC and another for GCDC.9,17 This could be a reflection of the difficulty in achieving reliable data at 25 °C or because modified γCDs interact slightly differently with BSs than seen with other CDs. A global compensation plot including data from the present and several other studies is in the Supporting Information. All values of ΔH° and ΔS° were higher for γCD complexes than for complexes with similar βCDs, which could be due to the larger contact area between solvent and γCD. This argument, however, is not supported by the ΔCp° data (presented below) or by the MD simulations. The change in heat capacity (ΔCp°) could be calculated from the linear slope of the fit (all regressions can be found in the Supporting Information) according to ΔC °p =

δ ΔH δT

Table 5. ΔASAnon per Substituent Molecule, Calculated from the Increment in ΔCp per Substituenta HP

SBE

Me

GC GDC GCDC

− (13.6) 17.8 (23.5) 20.4 (16.8)

33.4 (15.3) 22.0 (27.3) 23.5 (16.1)

9.9 (−) 13.6 (12.4) 14.5 (8.1)

a The numbers in parentheses are for modified βCDs and are also calculated from the increment in ΔCp per substituent. The ΔCp data for the modified βCDs are from the literature.12−14.

(2)

P

side chain was determined SBE side chains contributed most and methyl side chains the least to dehydration (Table 5). These experimentally derived values of ΔASAnon were verified by the MD simulations. The change in water accessible surface area (ΔASA) upon complexation was calculated from the ASA of the free and complexed species:

The hydration/dehydration of hydrophobic surface area is known to influence ΔCp°,11,38−41 where negative ΔCp° values are associated with dehydration of the hydrophobic area and vice versa. The ΔCp° values determined from eq 2 (Table 4) Table 4. Obtained Heat Capacity (ΔCp°) for the Investigated Complexesa

ΔASA = ASA Complex − (ASA CD,free + ASABS,free)

ΔCp° (J/mol/K) GC GDC GCDC

ΔASAnon per substituent (Å2/molecule)

γCD

SBE057

Me135

HP016

HP054

HP060

−277 −394 −370

−552 −574 −562

−469 −658 −653

−342.2 −432.8 −464.8

−304 −540 −554

−360 −541 −556

(3)

The changes in polar (ASApol) and nonpolar ASA (ASAnon) were calculated (Table 6). While the values of ΔASApol vary relatively little among the complexes some clear trends are observed for ΔASAnon. The values of ΔASAnon in GC complexes are consistently less negative than for complexes with GDC and GCDC. This agrees well with the experimental observation that the GC complexes have a less negative ΔCp°. Also listed in Table 6 is the dehydration per substituent, which may be compared to the experimental values in Table 5. Overall, the MD simulations support the conclusion that each substituent results in increased burial of nonpolar surface area in the order SBE > HP > Me. Considering the errors in estimating the values of ΔCp°, the uncertainties regarding the exact distribution of substituents, and the simple assumption of linearity between ΔCp° and ΔASAnon, there is a surprisingly

a The fitted graphs of ΔH° as function of temperature are found in the Supporting Information.

show that substitution leads to more negative values of ΔCp° in all cases. The precise value of ΔCp° depends on the size and DS of the substituent, with larger (SBE) and more numerous (Me) substituents having the most negative values. Regressions were made to investigate the dependence of ΔCp° on DS (see Supporting Information), but the fit to the HP-γCD-GC data 402


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Table 6. Calculated Polar and Nonpolar Water Accessible Surface Area (Å2) of Free Species and Complexes and the Change in Polar and Nonpolar ASA upon Complexation free species gCD mg138a mg138b HPg063 SBg063 GC

in complex with GC

ASApol

ASAnon

− − − − − 237

− − − − − 441 free species

ASApol gCD mg138a mg138b HPg063 SBg063 GDC

gCD mg138a mg138b HPg063 SBg063 GCDC a

ASAnon

− − − − − − − − − − 223 449 free species ASApol 774 464 475 754 1060 221

ASAnon 548 1113 1042 962 937 443

change in GC complex ΔASApol

ΔASAnon

contribution to ΔASAnon per substituenta

818 586 520 1137 547 1060 779 955 1077 885 − − in complex with GDC

−192 −181 −165 −213 −220 − change in GDC

−403 −418 −423 −448 −493 − complex

− −1 −2 −9 −18

ASApol

ΔASApol

ΔASAnon

contribution to ΔASAnon per substituenta

−419 −459 −424 −513 −534 − complex

− −4 0 −19 −23

ΔASAnon −426 −484 −455 −497 −554 −

contribution to ΔASAnon per substituenta − −5 −3 −14 −26

ASApol

ASAnon

ASAnon

813 578 538 1103 539 1067 788 898 1122 852 − − in complex with GCDC ASApol 794 518 528 784 1077 −

ASAnon 564 1072 1030 907 826 −

−184 −149 −159 −189 −161 − change in GCDC ΔASApol −200 −166 −167 −191 −203 −

This value is calculated as ΔASAnon divided by the number of substituents of the particular CD.

mg138a is used for “ASACD,free” in the calculation of ΔASAnon for the complexes with mg138b, the simulations show that each methyl substituent dehydrates 7−9 Å2 of hydrophobic surface, which is in better agreement with the experimental values derived from ΔCp°. Overall, the MD simulations support the interpretation that the observed increment in ΔCp° with an increased degree of substitution was caused by increased burial of hydrophobic surface as the substituents form hydrophobic contacts with the BSs. It is interesting to compare the present data with data for modified βCDs, for which similar observations have been made. In Table 5 the contribution to ΔASAnon per substituent are presented for a range of complexes between modified βCDs and BSs. The values are similar to those of the presently studied γCDs and show that the area dehydrated per substituent increases in the order Me < HP < SBE for the modified βCD. Overall, the substituents of γCDs had the same thermodynamic effect as when situated on βCDs.

good agreement between theory and experiment. For the complexes with GDC and GCDC, each HP-chain contributes an area of dehydration of 19 and 23 Å2 according to the MD simulations, while the values derived from ΔCp° predict 18 and 20 Å2. Likewise, for the complexes with GDC and GCDC the MD simulations show that each SBE chain contributes an area of dehydration of 14 and 26 Å2, while the experimental data predict 22 Å2 and 24 Å2. For the complexes with GC, no reliable experimental value could be obtained for the HP substituents and the area dehydrated by each SBE substituent seems unreasonable high. The complexes with Me-γCDs also pose a problem. Although the MD simulations predict that methyl substituents also cause an increased burial of hydrophobic surface, the values are much smaller than predicted from the experimental values of ΔCp°. Further analysis of the simulations reveal that the methyl groups at O3 all point inward toward the BS and therefore contribute more efficiently to the dehydration than the other methyl groups which either point outward (O2 methyls) or are located at the primary rim (O6 methyls). Thus, the reason for the small values of ΔASAnon for the simulated complexes with mg138a could be that only one O3 is methylated. mg138b has three methyl groups at O3 and has a larger hydrophobic contact surface with the BSs as reflected in the smaller values of ASAnon (Table 6). ASAnon for the uncomplexed mg138b is also somewhat smaller than for mg138a, which is due to the increased distortion of the macrocycle. Consequently, these two differences cancel out, so that the changes in ASAnon upon complexation (calculated according to eq 3), were similar for mg138a and mg138b, due to two opposing effects. It could be speculated that the simulations exaggerate the distortion of mg138b and that this is the main reason for the seemingly small dehydration area of the methyl substituents. If the value for the less distorted free

4. CONCLUSION The thermodynamics and structure of inclusion complexes between three biologically relevant BS and seven different γCDs were investigated. All complexes had 1:1 stoichiometry and the structures were very similar, indicating that substitution on γCDs did not affect the complex structure. The γCDs included the BS deeper than βCDs, because they included much of the steroid body, comprising the C-ring, D-ring and part of the B-ring. BS complexes with the natural γCD had the highest stability constants whereas Me300-γCD showed no detectable binding. Increasing the number of substituents on the CD had a negative effect on the stability constant, probably due to steric effects or distortion of the CD structure. This effect was more 403


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(6) Tan, X.; Lindenbaum, S. Studies on Complexation between βCyclodextrin and Bile Salts. Int. J. Pharm. 1991, 74, 127−135. (7) Cabrer, P. R.; Alvarez-Parrilla, E.; Al-Soufi, W.; Meijide, F.; Núñez, E. R.; Tato, J. V. Complexation of Bile Salts by Natural Cyclodextrins. Supramol. Chem. 2003, 15, 33−43. (8) Schönbeck, C.; Westh, P.; Madsen, J. C.; Larsen, K. L.; Städe, L. W.; Holm, R. Hydroxypropyl-Substituted β-Cyclodextrins: Influence of Degree of Substitution on the Thermodynamics of Complexation with Tauroconjugated and Glycoconjugated Bile Salts. Langmuir 2010, 26, 17949−17957. (9) Schönbeck, C.; Westh, P.; Madsen, J. C.; Larsen, K. L.; Städe, L. W.; Holm, R. Methylated β-Cyclodextrins: Influence of Degree and Pattern of Substitution on the Thermodynamics of Complexation with Tauro- and Glyco-Conjugated Bile Salts. Langmuir 2011, 27, 5832− 5841. (10) Ollila, F.; Pentikäinen, O. T.; Forss, S.; Johnson, M. S.; Slotte, J. P. Characterization of Bile Salt/Cyclodextrin Interactions using Isothermal Titration Calorimetry. Langmuir 2001, 17, 7107−7111. (11) Myers, J. K.; Pace, C. N.; Scholtz, J. M. Denaturant m Values and Heat Capacity Changes: Relation to Changes in Accessible Surface Areas of Protein Unfolding. Protein Sci. 1995, 4, 2138−2148. (12) Schönbeck, C.; Holm, R.; Westh, P.; Peters, G. H. Extending the Hydrophobic Cavity of β-Cyclodextrin Results in More Negative Heat Capacity Changes but Reduced Binding Affinities. J. Inclusion Phenom. Macrocyclic Chem. 2014, 78, 351−361. (13) Schönbeck, C.; Westh, P.; Holm, R. Complexation Thermodynamics of Modified Cyclodextrins: Extended Cavities and Distorted Structures. J. Phys. Chem. B 2014, 118, 10120−10129. (14) Holm, R.; Østergaard, J.; Schönbeck, C.; Jensen, H.; Shi, W.; Peters, G. H.; Westh, P. Determination of Stability Constants of Tauro- and Glyco-Conjugated Bile Salts with the Negatively Charged Sulfobutylether-β-Cyclodextrin: Comparison of Affinity Capillary Electrophoresis and Isothermal Titration Calorimetry and Thermodynamic Analysis of the Interaction. J. Inclusion Phenom. Macrocyclic Chem. 2014, 78, 185−194. (15) Holm, R.; Hartvig, R. A.; Nicolajsen, H. V.; Westh, P.; Østergaard, J. Characterization of the Complexation of Tauro- and Glyco-Conjugated Bile Salts with γ-cyclodextrin and 2-Hydroxypropylγ-Cyclodextrin using Affinity Capillary Electrophoresis. J. Inclusion Phenom. Mol. Recognit. Chem. 2008, 61, 161−169. (16) Cooper, A.; Nutley, M. A.; Camilleri, P. Microcalorimetry of Chiral Surfactant−Cyclodextrin Interactions. Anal. Chem. 1998, 70, 5024−5028. (17) Holm, R.; Schönbeck, C.; Askjær, S.; Westh, P. Thermodynamics of the Interaction of γ-Cyclodextrin and Tauro- and GlycoConjugated Bile Salts. J. Inclusion Phenom. Mol. Recognit. Chem. 2013, 75, 223−233. (18) Holm, R.; Madsen, J. C.; Shi, W.; Larsen, K. L.; Städe, L. W.; Westh, P. Thermodynamics of Complexation of Tauro- and GlycoConjugated Bile Salts with Two Modified β-Cyclodextrins. J. Inclusion Phenom. Mol. Recognit. Chem. 2011, 69, 201−211. (19) Barnes, S.; Geckle, J. M. High Resolution Nuclear Magnetic Resonance Spectroscopy of Bile Salts: Individual Proton Assignments for Sodium Cholate in Aqueous Solution at 400 MHz. J. Lipid Res. 1982, 23, 161−170. (20) Campredon, M.; Quiroa, V.; Thevand, A.; Allouche, A.; Pouzard, G. NMR Studies of Bile Acid Salts: 2D NMR Studies of Aqueous and Methanolic Solutions of Sodium Cholate and Deoxycholate. Magn. Reson. Chem. 1986, 24, 624−629. (21) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable Molecular Dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781− 1802. (22) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graphics 1996, 14, 33−38. (23) Guvench, O.; Greene, S. N.; Kamath, G.; Brady, J. W.; Venable, R. M.; Pastor, R. W.; Mackerell, A. D. Additive Empirical Force Field for Hexopyranose Monosaccharides. J. Comput. Chem. 2008, 29, 2543−2564.

pronounced for SBE and HP substituents than for methyl substituents. At 45 °C, complexation was mostly entropy driven for all complexes. Both ΔH° and ΔS° exhibited significant linear decreases with increasing temperature, as manifested in large negative values of ΔCp°. All substituents on the CDs caused a significant increase in ΔH°, ΔS° and -ΔCp°, depending on their number and type. HP substituents had a larger impact on ΔH° and ΔS° than SBE and methyl substituents, but profound enthalpy−entropy compensation was observed, so that the resulting effect on ΔG° was limited. The substituent-induced increments in ΔCp° could be quantitatively explained by an increased dehydration of the nonpolar surface area, as the substituents formed additional hydrophobic contacts between host (CD) and guest (BS). Each HP chain dehydrated 10−20 Å2 of BS surface area, the larger SBE side chains contributed the most to dehydration (22−33 Å2/substituent), and methyl side chains the least (10−15 Å2/substituent). The impact of the substituents on the complexation thermodynamics are thus very similar to that observed for complexation of BSs with modified β-CDs, and it is concluded that the quite different thermodynamic fingerprint of modified CDs, as compared to natural CDs, can be ascribed to increased burial of the hydrophobic BS surface.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b10536. Chemical structure of investigated bile salts, mass spectrometry and NMR spectra of investigated CDs, ROESY NMR spectra of complexes, stability constants and thermodynamic parameters for all complexes, extended thermodynamic analysis of experimental data, and dhydration surface area plots and calculations (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*(R.H.) E-mail: [email protected]. Telephone: (+45) 3643 3596. Fax: (+ 45) 3643 8242. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS David John Simpson is highly acknowledged for his valuable linguistic inputs. REFERENCES

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Hydration Differences Explain the Large Variations in the

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