Langmuir 1994,10, 1034-1039
1034
Formation of Inclusion Complexes of Cyclodextrins with Bile Salt Anions As Determined by NMR Titration Studies Z. J. Tan,+X. X. Zhu,X and G. R. Brown* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, PQ, Canada H3A 2K6 Received February 26, 1993. In Final Form: December 8,1 9 9 9 The interactionsbetween bile acid anions and cyclodextrins (CDs)in aqueous solution at room temperature have been studied in detail by the NMR titration method. The conformation adopted by the inclusion complex depends on the size of the cavity of the CD: The inclusion complexes with 8-CD result primarily from interactions with the smaller,hydrophilic side chains of the bile acid anions, while those with yCD, which has a larger cavity, involve interactions with the larger, hydrophobic end of bile acid anions. The results indicate that hydrophobic interactions between the host CD molecules and the guest bile acid anions are important in the formation of the complexes. Steric hindrance of certain groups, especially that due to the 12-OH group of the steroid skeleton,has important effects on the interaction. These results are consistent with additional information derived from molecular modeling studies.
Introduction Cyclodextrins (CDs) are macrocyclic oligosaccharides that have found important applications in molecular consisting recognition studies. Four different CDs (cu,@,r,s) of six, seven, eight, and nine glucosidicunits, respectively, have been isolated, and some of their physical properties are given in Table 1.l They possess relatively hydrophobic cavities, which have the profile of a truncated cone. They bind species of comparable size, generally forming 1:l inclusion complexes.'-' In recent years advantage has been taken of the complexation properties of CDs with a variety of molecules in developingchromatographic separation methods.- One of the best known examples is the reversed-phase HPLC method for the group separation of bile acids by adding solubleCDs to the mobile phase."12 A marked dependence of the capacity factors on the presence of a 12-OH group ' on the steroid skeleton was demonstrated.'l Reports of the interaction of CDs with bile acid anions are of interest since it is known that some functionalized
* To whom correspondence should be addressed.
Current address: Lash Miller Laboratories, Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1Al. $Current addrees: Department of Chemistry, Universitk de MontrBal,P.O.Box 6128, Station A, MontrBal,PQ,CanadaH3C 357. * Abstract published in Advance ACS Abstracts, February 15, f
1994. (1) Szejtli,J. Cyclodextn'n Technology;Kluwer AcademicPublishers: Dordrecht, Boston, London, 1988; Chapter 1. (2) Bender, M. L.; Komiyama, M. Cyclodeztrin Chemistry, Springer-Verlag: New York, 1978. (3) Saenger, W. Angew. Chem., Znt. Engl. Ed. 1980,19, 344. (4) Bender, H.In Advances in Biotechnological Processes; Mizrahi, A., Ed.; 1986, 6, 31. (5) For a review of the earlier work, see,e.g.: Szejtli, J. Cyclodextrin Technology;Kluwer Academic Publishers: Dordrecht, Boston, London, 1988, Chapter 7. (6) Isaaq, H.J.; McConnell, J. H.; Weiss, D. E.; Williams, D. G.; Saavedra, J. E. J. Liq. Chromatogr. 1986, 9, 1783. (7) h t r o n g ,D. W.; Yang,X.; Han, S. M.; Menges,R. A. Anal. Chem. 1987,69,2594. (8)Snopek, J.; Smolkova-Keulemansova, E.; Jelinek, I.; Dohnal, J.; Klinot, J.; Klinotova, E. J. Chromatogr. 1988,450, 373. (9) Shimada, K.; Maeue, T.; Toyoda, K.; Takani, M.; Nambara, T. J. Liq. Chromatogr. 1988,11, 1475. (10) Shimada, K.; Oe, T.; Kanno, C.; Nambara, T. Anal. Sci. 1988,4, 377. (11)Shimada, K.; Komine, Y.; Oe, T. J. Liq. Chromatogr. 1989, 12, 491. (12) Shimada,K.; Yoshida, H.; Komine, Y. J. Liq. Chromatogr. 1991, 14, 605.
0743-746319412410-l034$Q4.5O/O
18
21
OH R1 Cholic acid Glycocholic acid: Glycochenockoxycholicacid Lithocholic acid:
OH OH OH H
RZ
R3
OH OH H H
OH NHCHZCOOH NHCHZCOOH OH
Figure 1. Chemical structure of bile acids. Table 1. Selected Physical Properties of Cyclodextrind physical dimensions (A) no. of water solubility diameter CD glucoseunita (g/100mL) internal external a
6
B
7
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8
14.5 1.85 23.2
4.7-5.3 6.0-6.5 7.5-8.3
depth
14.6 i 0.4 7.9 i 0.1 15.4 i 0.4 7.9 i 0.1 17.5 f 0.4 7.9 i 0.1
CDs and their derivatives act as serum cholesterol level depressants.2 Bile acids are surfactant-like molecules that are used to assist in the digestion of fats by the formation of micelles and micellar aggregates. They possess a hydrophobic steroid skeleton to which are attached various hydrophilic groups, in particular the side chain carboxyl groups (Figure 1). Stereochemically the polar OH groups are located on one side of the steroid skeleton only, which imparb unique amphiphilic properties to them. Apparently bile acids interact also with drugs that are used for the treatment of hyperlipidemia, such as neomycin, clidamycin, kanamycin, and lincomycin,13 all having amine-substituted glucosidicstructural units and cleftlike structures, both of which are considered to be favorable in molecular re~ogniti0n.l~ Although the mechanism by which they decrease the serum cholesterol level is unclear, (13) Setchell, K. D. R.; Street, J . M.; Sjovall, J. In The Bile Acids, Setchell,K. D. R., Kritcheveky,D., Nair, P. P., Eds., Plenum Press: New York, London, 1988; Vol. 4, Chapter 12, p 543. (14) Rebek, J., Jr. Angew. Chem., Int. Ed. Engl. 1990,29, 245.
0 1994 American Chemical Society
Formation of Inclusion Complexes of Cyclodextrins
it has been suggested that they act as sequestrants for bile acid anions.13 The sequestering of bile acid anions in the gastrointestinal tract, by polymeric sorbents, is used clinically as a treatment to reduce serum cholesterol levels. This is known to reduce the incidence of cardiovascular diseases. Indeed, it has been established that lowering of plasma cholesterol levels actually results in the breakdown of plaque buildup on arterial walls.lS-l7 Unfortunately, currently available sorbents are relatively inefficient's20 so that an effective reduction of the cholesterol levels requires rather high daily doses (up to 54 g/day for the most commonly used sequestrant, cholestyramine). Obviously, this can cause discomfort to patients. Consequently, there is a great deal of interest in developing more effective sorbents for bile acids. Previous studies indicate that the low efficiency of the currently available sorbents results from a lack of specificity.21 Most are based on the ion exchange principle and show little discrimination toward different anions. To achieve a higher degree of binding specificity, it is desirable that the sequestrants have functional groups that can interact specificallywith bile acids. Since 8- and y-CDs have cavity sizes comparable with the size of bile acid anions, as demonstrated by the results from HPLC and molecular modeling, they were selected as candidates for studies of specific interactions with bile acids. The interactions between the host and guest molecules can be studied with a variety of techniques, in particular nuclear magnetic resonance (NMR) spectroscopy.2224The proton chemical shift changes of the interacting molecules can be monitored as a function of the molar concentration ratio. This method has been used previously in a number of studies of the complexation of CDs with a variety of organic molecules.23-21This paper presents the first study by NMR titration techniques of the interactions of 8- and y-CDs with bile acid anions in aqueous solutions. Experimental Section 8-CD (cycloheptaamylose),y CD (cyclooctaamylose),sodium glycocholate, and sodium glycochenodeoxycholate were purchased from Sigma Chemical Co.; sodium cholate and lithocholic acid were purchased from Aldrich; deuterated solvents (DzO) and reference materials were purchased from MSD Isotopes. The NMR experiments were performed at room temperature (21 f 1.5 "C) on a Varian XL-200 NMR spectrometer operating at 200 MHz for protons. The chemical shifts are considered to be accurate to about f0.0025 ppm. The magnetic field remained stable with the deuterium lock, the stability being tested with bile salt solutions in DzO with added references. To avoid interference with the binding, no internal reference was used for the interaction experiments. (15) Blankenhom, D. H. Can. J. Cardiol. 1989,5, 206. (16) Loacalzo, J. New Engl. J. Med. 1990,323, 1337. (17) Brown, G.; Albera, J. J.; Fisher, L. D.; Schaefer, S. M.; Lin, J.-T.; Kaplan, C.; Zhao, X.-Q.; Bisson, B. D.; Fitzpatrick, V. F.; Dodge, H. T. New England J. Med. 1990,323,1289. (18) Johns, W. H.; Bates, T. R. J. Pharm. Sci. 1969,58,178; 1970,59, 329; 1970,59,788. (19) Hageman, L. M.; Julow, D. A,; Schneider, D. L. Proc. SOC.Exp. Biol. Med. 1973, 143, 89. (20) Zhu, X. X.; Brown, G. R.; St-Pierre,L. E. J. Pharm. Sci. 1992,81, 65. (21) Koa, R.; White, J. L.; Hem, S. L.; Borin, M. T. Pharm. Res. 1991, 8, 238. (22) For a brief review, see Diederich, F. Angew. Chem., Znt. Engl. Ed. 1988,27, 362 and selected references cited therein. (23)Norwig, J.; Gelder, T.; Kraua, C.; Mehnert, W.; Rehse, K.; Fromming, K.-H. In Proceedings of the 4th International Symposium on Cyclodextrim; Huber, O., Szejtli, J., E&.; Kluwer Academic Publishers: Dordrecht, Boston, London, 1988; p 205. Taneya, A.; Yoahida, N.; Fujimoto, M. Zbid.; p 209; Vecchi, C.; Naggi,A.; Torri, G. Zbid.; p 215. (24) Guo, W.; Fung, B. M.; Christian, S. D. Langmuir 1992, 8, 446.
Langmuir, Vol. 10, No. 4, 1994 1035
CHOH
HI2 H7
H3
0.05
c
-0.05 0
I
I
I
O
I
I
I
40
20
I
I
I
60
c (g/l) Figure 3. Chemical shift changes of the methyl protons of cholate as a function of the concentration of cholate (diamonds, H12; circles, H18; squares, H19; triangles, H21). For the interaction studies, DzO solutionsof the bile acid anions containing various amounts of CD were prepared. The solutions were mixed and stored overnight at room temperature before the NMR spectra were recorded. To minimize intermolecular interactions, the concentrations of the bile acid anions were kept in the range 1.5-2.0 g/L, i.e., well below the critical micellar concentration (cmc). In the case of lithocholic acid, the solution of the guest was adjusted to p[H+Dl 11-12, by adding NaOH, until all of the lithocholic acid was dissolved. Aminated &CD was prepared by reacting 8-CD with chloroethylamine in water at room temperature overnight under the catalysis of a base.' The ethylamine group was attached to the C2 position on the sugar. The interactions of the host-guest complexds were modeled with the molecular modeling program QUANTA 1of Polygon on a VAX computer.
Results and Discussion In the NMR titration experimentsattention was focused mainly on the chemical shift changes of the methyl protons at positions 18,19, and 21 (H18, H19, H21) of the bile acid anions and, in certain cases, some other protons (such as H12, H3, and H7) for which the resonance peaks are well resolved in the lH NMR spectrum (Figure 2). Intermolecular Interaction of Bile Acid Anions. Even in the absence of added CDs, the chemical shifts of the methyl protons H12, Hl8, and H19 exhibit relatively large downfield changes with the increase in the concentration of cholate in DzO (Figure 3). Significant changes in the chemical shifts are detected at a concentration of about 6g/L. On the basis of previouslyreported cmc values in the vicinity of 4.3-5.7 g/L,26*2s this corresponds to the (25) Small, D. M. In The Bile Acids, Nair, P. P., Kritchevsky, D., W.; Plenum Press: New York, 1971; Vol. 1, Chapter 8, p 249.
1036 Langnuir, Vol. 10, No. 4, 1994
e 0.10
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Tan et al.
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Figure 5. Molecular model of the complexof cholate with 8-CD.
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' ' ' ' I ' ' ' ' I ' ' ' ' H/G Molar Ratio Figure 4. Complexation-inducedchanges in chemical shifts of the methyl protons of the bile acids as a function of the host: guest molar ratio (host 8-CD): (A) guest cholate, 2.81 mM (B) guest glycocholate,3.653 m M (C)guest glycochenodeoxycholate, 3.673 mM; (D) guest lithocholate, 4.810 mM, p[D+Hl 11-12; circles, H18; squares, H19; triangles, H21. onset of micelle formation. The protons at positions H12, H18, and H19, which are either a part of the steroid skeleton or directly attached to it, are most affected by these intermolecular interactions. Only small changes are observed in the chemical shifts of protons H21 and H7, upfield and downfield, respectively. In keeping with the well-known back-to-back interaction of bile acids at concentrations exceeding the cmc, there is no change in the chemical shift of proton H3. This is conformed by a concomitant change of the shape of methylene-methine "envelope" multiplets. Assuming that the changes in the chemical shifts correspond to the formation of the micelles and clusters -0.10
' ' ' '
I
of micelles,calculations based on the conjugated primarysecondary micellar formation model2sn yield an aggregation number of 2.1 and formation constants of 74 and 26, for primary micelles and for the secondary micellar aggregates,respectively. These results are consistent with the previously reported values obtained from light scattering measurement.a.27 Interactions of the Bile Acid Anions with 8Cyclodextrin. The titration curves of various bile acid anions with the B-CD, shown in Figure 4, indicate the formation of 1:l inclusion complexes at the final stage. While both the 12-OH and the side chain on the bile acid affect the complexation between the CD and the bile acid, the former has a much stronger effect than the latter. For the addition of cholate (Figure 4A) or glycocholate (Figure 4B) to 8-CD the effect of an increase in concentration on the changes in the chemical shifts of protons H18 and H19 obviously differs from those that result from the intermolecular interactions, Le., micelle formation. Protons H18 and H21 (a doublet in the spectrum) both have larger downfield changes in the chemical shift than proton H19, in keeping with the formation of an inclusion complex which has the side chain of the bile acid anion located within the cavity of 8-CD, to yield a complex of the form shown by the molecular model in Figure 5. By contrast, only a small change (ca. +0.03 ppm) in the chemical shift of the H21 protons results from the addition of glycochenodeoxycholate to 8-CD (Figure 4C), much smaller than that of the H19 protons (ca. +0.07 ppm). A strong interaction accompanies the formation of the inclusion complex and results in the appearance of a new peak for the H18 protons at a chemical shift 0.113 ppm downfield from the original peak (Figure 6). When glycochenodeoxycholate forms the complex, it apparently penetrates the cavity of the B-CD to a greater extent than either cholate or glycocholate. Indeed, the small change of the chemical shift of the H21 protons, in spite of the strong binding, suggests that the methyl group at position 21 protrudes through the cavity. The chemical shifts of protons H18 and H19 of lithocholate show similar large downfield changes, ca. +0.11 ppm (Figure 4D), that increase gradually, with an increase in concentration. By comparison, the H21 protons experience small upfield chemical shift changes that pass through a minimum, ca. -0.05 ppm, a t a hoskguest molar ratio of 1and then return to zero as the host concentration increases. It is also of interest to note that the addition of 8-CD to lithocholate causes the overlapped resonance (26) Conte, G.; Blasi, R. D.; Giglio, E.; Parretta, A.; Pawl, N. V. J. Phys. Chem. 1984,88,5720. (27) Mazer, N. A.; Carey, M. C.; Kwamick, R. F.; Benedek, G. B. Biochemistry 1979,18, 3064.
Formation of Inclusion Complexes of Cyclodextrins 1.2
Langmuir, Vol. 10, No. 4,1994 1037
1
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0 0
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4 L
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HIG Molar Ratio
Figure 6. A plot of the area ratio of the complexation-induced peak and the original peak of the Hl8 methyl protpns of glycochenodeoxycholate as a function of the hoskguest molar ratio (host j3-CD; guest glycochenodeoxycholate, 3.673 mM).
peaks for protons H19 and H21 to separate to produce a doublet for the H21 protons so that the NMR peaks can be assigned easily. These results indicate that in the complex lithocholate is located even deeper in the cavity than glycochenodeoxycholate. (In this case there may also be a pH effect since the solution is more basic than the other bile salt solutions.) These results can be used to develop a rational explanation for the effect of added j3-CD on the separation of bile acids by HPLC. According to Shimada et al.,11J2the HPLC capacity factors of the bile saltswith a l2a-hydroxyl group are not influenced significantly by the addition of j3-CD while those of the other bile acids are sharply decreased. Molecular modeling of the host-guest interaction predicts that the polarity and bulkiness of the 12-OH group, shown for the cholate ion (Figure 5 ) , give rise to a significant energy barrier for the entry of bile acids into the j3-CD cavity. For the bile acid anions that do not have the 12-OH,e.g., glycochenodeoxycholate, this effect is not seen, even in the presence of the 7-OH group. This is in keeping with the formation of a more stable complex with the bile acid anion located well within the j3-CD cavity. This sharply decreases the HPLC capacity factors. Since lithocholate has neither a 12-OHnor 7-OH and, therefore, has less steric hindrance, this phenomenon becomes more obvious. Interactions of the Bile Acid Anions with 7Cyclodextrin. The NMR titration studies of the interactions of the bile acid anions with y C D , which has a larger cavity than 8-CD, also indicate the formation of 1:l complexes (Figure 7). Neither the 12-OH nor the side chain of the bile acid anion seems to have a significant effect on the interactions, according to the chemical shift changes of the H19 protons. Thus, the interactions differ somewhat from those with D-CD. The addition of y C D to cholate results in an upfield shift of the signal of the H18 protons (Figure 7A), but the change is much smaller than in the case of added j3-CD (Figure 4A). The H19 protons have larger, but opposite (downfield), changes. The proton signal of H21 overlaps with that of H19 and shows no change in chemical shift upon addition of yCD. Similar behavior is seen in the case of glycocholate (Figure 7B). At low hoskguest molar ratios, the proton peaks of H18 and H21 both have an upfield and then a downfield change, but then both return to their original chemical shifts at higher hoskguest ratios. Their changes are smaller than that of H19. These changes
0
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H/G Molar Ratio Figure 7. Complexation-induced changes in chemical shifts of the methyl protons of the bile acids as a function of the host: guest molar ratio (host 7-CD): (A) guest cholate, 2.392 m M (B) guestglycocholate,3.653mM; (C) guest glycochenodeoxycholate, 3.673 mM; circles, H18; squares, H19; triangles, H21.
are in keeping with an interaction of the 7-CD with the hydrophobic end of the bile acid anions. As a result of these interactionsthe proton peaks are broadened at higher host:guest molar ratios. The H21 protons of glycochenodeoxycholate also have small upfield changes (ca. 0.03-0.04 ppm) initially and then return to the original position at higher hoskguest molar ratios (Figure 7C). The peaks of the protons H18 and H19 have up- and downfield changes, respectively, of similar magnitude. The similarity of these results to those for glychocholate indicates that the 12-OH on the steroid skelton has a minimal effect on the host-guest interaction. Again, it would seem that the absence of the 12-OH group permits the glycochenodeoxycholate to penetrate further into the cavity of yCD, and as a consequence the overlapped peaks of the protons H19 and H21 separate. It is of interest to note that for the complexation of bile acid anions with 7-CD the only significant chemical shift change takes place for the H19 protons which are located closer to the hydrophobic, steroid portion. This suggests that the complexed bile acid anions reside more deeply within the CD cavity, so that the hydrophobicinteractions are maximized. Alternatively, the complexed anions are oriented so that the larger hydrophobic steroid skeleton lies within the cavity while the side chain protrudes from
Tan et al.
1038 Langmuir, Vol. 10, No. 4, 1994 Table 2. Formation Constants (M-1) for Cyclodextrin-Bile Acid Inclusion Complexes. CA GCA GDCA LCA 8-CD 1488 336 371 886 Y-CD 362 210 239 a Key: CD, cyclodextrin;CA, cholate;GCA, glycocholate;GDCA, glycochenodeoxycholate;LCA, lithocholate. b p[H+Dl 11-12.
the larger end. This is discussed further in connection with the molecular modeling results, which show that the relative sizes permit such an orientation. Interactions of Bile Acids with Aminated 8Cyclodextrin. Upon interaction of cholatewith aminated &CD, the resonances for the H18, H19, and H21 protons experience downfield shifts of ca. 0.05 ppm, while the changes for the protons H18 and H21 are larger (0.13-0.15 ppm). These chemicalshift changes are, in general, similar to but more pronounced than those for the interaction with 8-CD. The amino group contributes favorably to the complexation by the additional electrostatic interactions between the bile acid anions and 8-CD. Complex Formation Constants. The formation of 1:l inclusion complexes of the host molecule, H, and the guest molecule, G, can be expressed as
H+G+HG
(1)
Therefore, the stoichiometric formation constant is given by
K = [HGI/[Hl [GI
(2)
Assuming that the chemical shift (6) of a proton signal of the guest molecule can be represented by the sum of fractional contributions from the free and complexed guest molecules, then
(3) where [HI0 and [GI0 are the initial concentrations of the host and guest, respectively. Then, the chemical shift change, A = 6 - 6 ~ can , be represented by
where & = ~ H G 6 ~ .Substitution of [HG] from eq 4 into eq 3 and taking into account that, for 1:l complexes, [HI = [HI0 - [HGI and [GI = [GI0 - [HGI, yields
Thus, the formation constants for the complexes can be calculated by curve fitting of the proton chemical shift changes. Values for [Hlo, [Glo, and A were obtained directly from the experimentaldata. A first approximation of Ao, based on the value of A at the highest concentrations, was used in a curve fitting routine involving eq 5, to obtain precise values of & and K. These calculations were based on the chemical shift changes of H18, except in the case of glycochenodeoxycholate (for which the chemical shift change of H19 was used). The resulting thermodynamic constants for the formation of the inclusion complexes, given in Table 2, indicate a stronger interaction of both
8- and yCDs with cholate than with either glycocholate or glycochenodeoxycholate. Since the conjugation with glycine renders the bile acid anions more hydrophilic,these results are consistent with a hydrophobic interaction. Molecular Modeling of the Interaction. According to the molecular modeling studies, the internal diameters of 8-CD and 7-CD are 6.0-6.5 and 7.5-8.3 A, respectively (Table 1). The length of the steroid portion of the bile acid anion is ca. 7-8 A. For the bile acid anions lacking the 12-OH group, e.g., glycochenodeoxycholate and lithocholate, the smaller hydrophilic end has a maximum diameter of 6.0 f 0.5 A, while the maximum diameter of the larger hydrophobic portion is 7.4 f 0.5 A. By comparison, for the bile acid anions having the 12-OH group, e.g., cholate and glycocholate, the diameter of the hydrophobic end increases by 0.6-1.0 A. On the basis of these dimensions, steric considerations suggest that in spite of the importance of hydrophobic interactions the bile acid anions can only penetrate the &CD cavity via the hydrophilic end, which is supported by the NMR titration results. When the diameter of the cavity is increased, as in the case of yCD, it becomes similar in size to the steroid skeleton so the bile acid anions can enter further into the cavity. As a result, more of the hydrophobic skeleton will be affected, as is seen from the NMR chemical shift changes. The relative sizes of the host and guest even permit entry of the bile acid anions into the cavity via the larger hydrophobic end. It is safe to say that in this case the smaller hydrophilic end of the bile salt guests can protrude through the CD cavity to maximize the hydrophobic interactions. The NMR results indicatethat the inclusion complex predominantlyinvolves interactions with the bulky hydrophobic steroid skeleton. In either case, the 12-OH is the first large polar group that is encountered as the bile acid anions enter the cavity, and it has an important effect on the interaction, as evidenced by the fact that it causes the differences in the proton NMR chemical shift changes that result upon complexation with 8-CD. This effect is further evidenced by the fact that glycochenodeoxycholateis able to penetrate the cavity of 8-CD further than can glycocholate or cholate. The presence of 7-OH also affects the complexation of the bile acid anions with CDs, as shown by an even deeper penetration of the 8-CD cavity by lithocholate than by glycochenodeoxycholate. This is in agreement with the theory that when a molecule interacts with CDs hydrophobic interactions will take priority unless steric hindrance is a major concern.28 The HPLC results reported previously by other researchers can also be explained on the basis of this interaction mechanism. The binding between the host and guest molecules, due to van der Waals interactions and hydrogen bonding, is accompaniedby the release of high-energywater molecules due to complex formation and the release of the strain energy in the macromolecular ring of CD. The significant effect of the 12-OH group on the interaction indicates that the main contribution of the binding force comes from hydrophobic interactions, taking note that the cavity of the CDs is also rather hydrophobic in nature. Conclusions The main drivingforce for the formation of the inclusion complexes of bile acid anions with CDs is hydrophobic interactions. The NMR titration results show that the formation of the complexes depends on the cavity size of (28) Arnold, E. N.; Lillie, T.S.;Beesley, T.E.J. Liq. Chromotogr. 1989, 12, 337.
Formation of Inclusion Complexes of Cyclodextrins the CD. The bile acid anions generally penetrate the cavity of p-CD via the smaller, hydrophilic side chain led by the carboxyl group. Polar hydroxyl g r o u p at positions 12 and 7 on the steroid skeleton have a marked effect on the complexation with &CD. The modification of the hydrophobicity of the bile acid anion, by conjugation with glycine, also affects the interactions. The NMR results indicate that the complexes with 7-CD involve a deeper penetration into the CD cavity. Both the hydroxyl and
Langmuir, Vol. 10, No. 4, 1994 1039 side chain groups have a relatively small effect on the complexation of yCD.
Acknowledgment. Financial support in the form of operating granta from the Natural Sciences and Engineering Research Council (NSERC) of Canada and from the Qu6bec Government (Fonds FCAR) is gratefully acknowledged. In addition, Z.J.T. would like to acknowledge, with thanks, financial support from CIDA.
